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Lefkowitz, Robert J.

Overview:

The focus of work in this laboratory is on the elucidation of the molecular properties and regulatory mechanisms controlling the function of G protein-coupled receptors. As model systems we utilize the so called adrenergic receptors for adrenaline and related molecules. The goal is to learn the general principles of signal transduction from the outside to the inside of the cell which are involved in systems as diverse as sensory perception, neuro- transmitter and hormonal signaling. Studies are performed with isolated protein, whole cells in culture and even in vivo in whole animals.

Current projects emphasize attempts to understand regulation of the receptors and their desensitization which occurs in response to persistent stimulation. We are isolating the enzymes and proteins involved in these processes and studying their mechanisms of action in isolated protein and cellular systems as well as in whole animals. Most important are special enzymes called G protein-coupled receptor kinases which phosphorylate the receptors and lead to their desensitization which occurs when they bind a second protein called barrestin. Most recently we have been developing lines of transgenic animals in which these various proteins are either overexpressed or "knockedout" by homologous recombination. These genetically altered animal lines are helping to shed new light on the ways in which receptors are regulated. They also have suggested several novel approaches to human therapeutics.

While no clinical trials are currently in progress in our program we are experimenting with novel approaches to the treatment of congestive heart failure in animal models. Specifically we are injecting recombinant adenovirus encoding either the b-adrenergic receptors or inhibitors of the b-adrenergic receptor kinase down the coronary arteries of rabbits. The hope is that these gene products when expressed in the myocardium will markedly enhance cardiac contractility.

Dr. Lefkowitz has received a great deal of recognition for his research including election to the National Academy of Sciences and the Institute of Medicine of the National Academy of Sciences as well as the receipt of numerous awards. Most recently these have included the The Louis and Artur Lucian Award for Research in Circulatory Disease, The Fred Conrad Koch Award - The Endocrine Society, The 2001 Jessie Stevenson Kovalenko Medal - The National Academy of Sciences and The Peter Harris Distinguished Scientist Award, International Society of Heart Research. Dr. Lefkowitz writes numerous review articles in the areas of hormone and drug receptors and their regulation and is a consultant for several drug companies which specialize in drugs which may affect signal transduction processes such as Norak, Lexicon Genetics and Genentech.

Positions:

James B. Duke Professor of Medicine

Medicine, Cardiology
School of Medicine

Professor of Medicine

Medicine, Cardiology
School of Medicine

Professor of Pathology

Pathology
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Professor of Chemistry

Chemistry
Trinity College of Arts & Sciences

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1966

M.D. — Columbia University

News:

Awards:

Nobel Prize-Chemistry. Nobel Foundation.

Type
National
Awarded By
Nobel Foundation
Date
January 01, 2012

Life Science and Medicine. Shaw Prize Foundation, The.

Type
National
Awarded By
Shaw Prize Foundation, The
Date
January 01, 2007

National Medal of Science. National Science and Technology Medals Foundation.

Type
National
Awarded By
National Science and Technology Medals Foundation
Date
January 01, 2007

Herbert Tabor/Journal of Biological Chemistry Lectureship. American Society for Biochemistry and Molecular Biology.

Type
National
Awarded By
American Society for Biochemistry and Molecular Biology
Date
January 01, 2004

Established Professional/Walter B. Cannon Award Lecture. American Physiological Society.

Type
National
Awarded By
American Physiological Society
Date
January 01, 2001

Jessie Stevenson Kovalenko Medal. National Academy of Science.

Type
National
Awarded By
National Academy of Science
Date
January 01, 2001

Endocrinology and Metabolism Section Awards/Solomon A. Berson Distinguished Lecture. American Physiological Society.

Type
National
Awarded By
American Physiological Society
Date
January 01, 1996

Member. Institute of Medicine of The National Academies.

Type
National
Awarded By
Institute of Medicine of The National Academies
Date
January 01, 1994

Award for Distinguished Research in the Biomedical Sciences. Association of American Medical Colleges.

Type
National
Awarded By
Association of American Medical Colleges
Date
January 01, 1990

Fellows. American Academy of Arts and Sciences.

Type
National
Awarded By
American Academy of Arts and Sciences
Date
January 01, 1988

Gairdner Award. Gairdner Foundation, The.

Type
National
Awarded By
Gairdner Foundation, The
Date
January 01, 1988

Members/ Foreign Associates. National Academy of Science.

Type
National
Awarded By
National Academy of Science
Date
January 01, 1988

Investigator/Alumni Investigator. Howard Hughes Medical Institute.

Type
National
Awarded By
Howard Hughes Medical Institute
Date
January 01, 1976

Publications:

Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis.

β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-βarr conformations can carry out distinct functions.

Authors
Cahill, TJ; Thomsen, ARB; Tarrasch, JT; Plouffe, B; Nguyen, AH; Yang, F; Huang, L-Y; Kahsai, AW; Bassoni, DL; Gavino, BJ; Lamerdin, JE; Triest, S; Shukla, AK; Berger, B; Little, J; Antar, A; Blanc, A; Qu, C-X; Chen, X; Kawakami, K; Inoue, A; Aoki, J; Steyaert, J; Sun, J-P; Bouvier, M; Skiniotis, G; Lefkowitz, RJ
MLA Citation
Cahill, TJ, Thomsen, ARB, Tarrasch, JT, Plouffe, B, Nguyen, AH, Yang, F, Huang, L-Y, Kahsai, AW, Bassoni, DL, Gavino, BJ, Lamerdin, JE, Triest, S, Shukla, AK, Berger, B, Little, J, Antar, A, Blanc, A, Qu, C-X, Chen, X, Kawakami, K, Inoue, A, Aoki, J, Steyaert, J, Sun, J-P, Bouvier, M, Skiniotis, G, and Lefkowitz, RJ. "Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis." Proceedings of the National Academy of Sciences of the United States of America 114.10 (March 2017): 2562-2567.
PMID
28223524
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
114
Issue
10
Publish Date
2017
Start Page
2562
End Page
2567
DOI
10.1073/pnas.1701529114

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Authors
Ahn, S; Kahsai, AW; Pani, B; Wang, Q-T; Zhao, S; Wall, AL; Strachan, RT; Staus, DP; Wingler, LM; Sun, LD; Sinnaeve, J; Choi, M; Cho, T; Xu, TT; Hansen, GM; Burnett, MB; Lamerdin, JE; Bassoni, DL; Gavino, BJ; Husemoen, G; Olsen, EK; Franch, T; Costanzi, S; Chen, X; Lefkowitz, RJ
MLA Citation
Ahn, S, Kahsai, AW, Pani, B, Wang, Q-T, Zhao, S, Wall, AL, Strachan, RT, Staus, DP, Wingler, LM, Sun, LD, Sinnaeve, J, Choi, M, Cho, T, Xu, TT, Hansen, GM, Burnett, MB, Lamerdin, JE, Bassoni, DL, Gavino, BJ, Husemoen, G, Olsen, EK, Franch, T, Costanzi, S, Chen, X, and Lefkowitz, RJ. "Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library." Proceedings of the National Academy of Sciences of the United States of America 114.7 (February 2017): 1708-1713.
PMID
28130548
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
114
Issue
7
Publish Date
2017
Start Page
1708
End Page
1713
DOI
10.1073/pnas.1620645114

GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Authors
Thomsen, ARB; Plouffe, B; Cahill, TJ; Shukla, AK; Tarrasch, JT; Dosey, AM; Kahsai, AW; Strachan, RT; Pani, B; Mahoney, JP; Huang, L; Breton, B; Heydenreich, FM; Sunahara, RK; Skiniotis, G; Bouvier, M; Lefkowitz, RJ
MLA Citation
Thomsen, ARB, Plouffe, B, Cahill, TJ, Shukla, AK, Tarrasch, JT, Dosey, AM, Kahsai, AW, Strachan, RT, Pani, B, Mahoney, JP, Huang, L, Breton, B, Heydenreich, FM, Sunahara, RK, Skiniotis, G, Bouvier, M, and Lefkowitz, RJ. "GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling." Cell 166.4 (August 3, 2016): 907-919.
PMID
27499021
Source
epmc
Published In
Cell
Volume
166
Issue
4
Publish Date
2016
Start Page
907
End Page
919
DOI
10.1016/j.cell.2016.07.004

Alfred Goodman Gilman (1941-2015).

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Alfred Goodman Gilman (1941-2015)." Nature 529.7586 (January 2016): 284-.
PMID
26791713
Source
epmc
Published In
Nature
Volume
529
Issue
7586
Publish Date
2016
Start Page
284
DOI
10.1038/529284a

beta-arrestin2 Is Necessary for Development of MPLW515L Mutant Primary Myelofibrosis

Authors
Rein, LAM; Wisler, JW; Theriot, BS; Huang, L-Y; Premont, RT; Lefkowitz, RJ
MLA Citation
Rein, LAM, Wisler, JW, Theriot, BS, Huang, L-Y, Premont, RT, and Lefkowitz, RJ. "beta-arrestin2 Is Necessary for Development of MPLW515L Mutant Primary Myelofibrosis." December 3, 2015.
Source
wos-lite
Published In
Blood
Volume
126
Issue
23
Publish Date
2015

The role of β-arrestin2-dependent signaling in thoracic aortic aneurysm formation in a murine model of Marfan syndrome.

Ang II type 1a receptor (AT1aR)-mediated activation of MAPKs contributes to thoracic aortic aneurysm (TAA) development in Marfan syndrome (MFS). β-Arrestin2 (βarr2) is known to mediate AT1aR-dependent MAPK activation, as well as proproliferative and profibrotic signaling in aortic vascular smooth muscle cells. Therefore, we investigated whether βarr2-dependent signaling contributes to TAA formation in MFS. We used a murine model of MFS [fibrillin (Fbn)(C1039G/+)] to generate an MFS murine model in combination with genetic βarr2 deletion (Fbn(C1039G/+)/βarr2(-/-)). Fbn(C1039G/+)/βarr2(-/-) mice displayed delayed aortic root dilation compared with Fbn(C1039G/+) mice. The mRNA and protein expression of several mediators of TAA formation, including matrix metalloproteinase (MMP)-2 and -9, was reduced in the aorta of Fbn(C1039G/+)/βarr2(-/-) mice relative to Fbn(C1039G/+) mice. Activation of ERK1/2 was also decreased in the aortas of Fbn(C1039G/+)/βarr2(-/-) mice compared with Fbn(C1039G/+) animals. Small interfering RNA targeting βarr2 inhibited angiotensin-stimulated expression of proaneurysmal signaling mediators in primary aortic root smooth muscle cells. Angiotensin-stimulated expression of the proaneurysmal signaling mediators MMP-2 and -9 was inhibited by blockade of ERK1/2 or the EGF receptor, whereas blockade of the transforming growth factor-β receptor had no effect. These results suggest that βarr2 contributes to TAA formation in MFS by regulating ERK1/2-dependent expression of proaneurysmal genes and proteins downstream of the AT1aR. Importantly, this demonstration of the unique signaling mechanism by which βarr2 contributes to aneurysm formation identifies multiple novel, potential therapeutic targets in MFS.

Authors
Wisler, JW; Harris, EM; Raisch, M; Mao, L; Kim, J; Rockman, HA; Lefkowitz, RJ
MLA Citation
Wisler, JW, Harris, EM, Raisch, M, Mao, L, Kim, J, Rockman, HA, and Lefkowitz, RJ. "The role of β-arrestin2-dependent signaling in thoracic aortic aneurysm formation in a murine model of Marfan syndrome." American journal of physiology. Heart and circulatory physiology 309.9 (November 2015): H1516-H1527.
PMID
26371162
Source
epmc
Published In
American journal of physiology. Heart and circulatory physiology
Volume
309
Issue
9
Publish Date
2015
Start Page
H1516
End Page
H1527
DOI
10.1152/ajpheart.00291.2015

Inspiring the next generation of physician-scientists.

As academic physician-scientists, one of the most important things we do is mentor young trainee-scientists. There obviously is no one right way to mentor or a set of rules one can follow; it's a very personal matter, and very much depends on one's personality. For much of my career, I gave very little thought as to how I mentored my trainees or to whether I was any good at it. Like many investigators, perhaps, I was just too busy with the daily activities of research to consider how I was guiding my students. Here, I take a look back and reflect on my experiences as a mentor and the factors that I believe contribute to the success of trainees as independent scientists.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Inspiring the next generation of physician-scientists." The Journal of clinical investigation 125.8 (August 3, 2015): 2905-2907.
PMID
26237039
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
8
Publish Date
2015
Start Page
2905
End Page
2907
DOI
10.1172/jci83222

N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.

Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

Authors
Soto, AG; Smith, TH; Chen, B; Bhattacharya, S; Cordova, IC; Kenakin, T; Vaidehi, N; Trejo, J
MLA Citation
Soto, AG, Smith, TH, Chen, B, Bhattacharya, S, Cordova, IC, Kenakin, T, Vaidehi, N, and Trejo, J. "N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias." Proceedings of the National Academy of Sciences of the United States of America 112.27 (July 2015): E3600-E3608.
PMID
26100877
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
27
Publish Date
2015
Start Page
E3600
End Page
E3608
DOI
10.1073/pnas.1508838112

Allosteric modulation of β-arrestin-biased angiotensin II type 1 receptor signaling by membrane stretch.

It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-biased ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-biased signaling remains unknown. Implicit in the concept of biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.

Authors
Tang, W; Strachan, RT; Lefkowitz, RJ; Rockman, HA
MLA Citation
Tang, W, Strachan, RT, Lefkowitz, RJ, and Rockman, HA. "Allosteric modulation of β-arrestin-biased angiotensin II type 1 receptor signaling by membrane stretch." The Journal of biological chemistry 289.41 (October 2014): 28271-28283.
PMID
25170081
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
41
Publish Date
2014
Start Page
28271
End Page
28283
DOI
10.1074/jbc.m114.585067

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Authors
Shukla, AK; Westfield, GH; Xiao, K; Reis, RI; Huang, L-Y; Tripathi-Shukla, P; Qian, J; Li, S; Blanc, A; Oleskie, AN; Dosey, AM; Su, M; Liang, C-R; Gu, L-L; Shan, J-M; Chen, X; Hanna, R; Choi, M; Yao, XJ; Klink, BU; Kahsai, AW; Sidhu, SS; Koide, S; Penczek, PA; Kossiakoff, AA; Woods, VL; Kobilka, BK; Skiniotis, G; Lefkowitz, RJ
MLA Citation
Shukla, AK, Westfield, GH, Xiao, K, Reis, RI, Huang, L-Y, Tripathi-Shukla, P, Qian, J, Li, S, Blanc, A, Oleskie, AN, Dosey, AM, Su, M, Liang, C-R, Gu, L-L, Shan, J-M, Chen, X, Hanna, R, Choi, M, Yao, XJ, Klink, BU, Kahsai, AW, Sidhu, SS, Koide, S, Penczek, PA, Kossiakoff, AA, Woods, VL, Kobilka, BK, Skiniotis, G, and Lefkowitz, RJ. "Visualization of arrestin recruitment by a G-protein-coupled receptor." Nature 512.7513 (August 2014): 218-222.
Website
http://hdl.handle.net/10161/13107
PMID
25043026
Source
epmc
Published In
Nature
Volume
512
Issue
7513
Publish Date
2014
Start Page
218
End Page
222
DOI
10.1038/nature13430

Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR).

The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.

Authors
Strachan, RT; Sun, J-P; Rominger, DH; Violin, JD; Ahn, S; Rojas Bie Thomsen, A; Zhu, X; Kleist, A; Costa, T; Lefkowitz, RJ
MLA Citation
Strachan, RT, Sun, J-P, Rominger, DH, Violin, JD, Ahn, S, Rojas Bie Thomsen, A, Zhu, X, Kleist, A, Costa, T, and Lefkowitz, RJ. "Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR)." The Journal of biological chemistry 289.20 (May 2014): 14211-14224.
PMID
24668815
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
20
Publish Date
2014
Start Page
14211
End Page
14224
DOI
10.1074/jbc.m114.548131

Recent developments in biased agonism

The classic paradigm of G protein-coupled receptor (GPCR) activation was based on the understanding that agonist binding to a receptor induces or stabilizes a conformational change to an 'active' conformation. In the past decade, however, it has been appreciated that ligands can induce distinct 'active' receptor conformations with unique downstream functional signaling profiles. Building on the initial recognition of the existence of such 'biased ligands', recent years have witnessed significant developments in several areas of GPCR biology. These include increased understanding of structural and biophysical mechanisms underlying biased agonism, improvements in characterization and quantification of ligand efficacy, as well as clinical development of these novel ligands. Here we review recent major developments in these areas over the past several years. © 2013 .

Authors
Wisler, JW; Xiao, K; Thomsen, ARB; Lefkowitz, RJ
MLA Citation
Wisler, JW, Xiao, K, Thomsen, ARB, and Lefkowitz, RJ. "Recent developments in biased agonism." Current Opinion in Cell Biology 27.1 (April 1, 2014): 18-24. (Review)
PMID
24680426
Source
scopus
Published In
Current Opinion in Cell Biology
Volume
27
Issue
1
Publish Date
2014
Start Page
18
End Page
24
DOI
10.1016/j.ceb.2013.10.008

Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.

The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.

Authors
Staus, DP; Wingler, LM; Strachan, RT; Rasmussen, SGF; Pardon, E; Ahn, S; Steyaert, J; Kobilka, BK; Lefkowitz, RJ
MLA Citation
Staus, DP, Wingler, LM, Strachan, RT, Rasmussen, SGF, Pardon, E, Ahn, S, Steyaert, J, Kobilka, BK, and Lefkowitz, RJ. "Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies." Mol Pharmacol 85.3 (March 2014): 472-481.
PMID
24319111
Source
pubmed
Published In
Molecular pharmacology
Volume
85
Issue
3
Publish Date
2014
Start Page
472
End Page
481
DOI
10.1124/mol.113.089516

beta-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Authors
Simard, E; Kovacs, JJ; Miller, WE; Kim, J; Grandbois, M; Lefkowitz, RJ
MLA Citation
Simard, E, Kovacs, JJ, Miller, WE, Kim, J, Grandbois, M, and Lefkowitz, RJ. "beta-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration." PLOS ONE 8.11 (November 8, 2013).
PMID
24255721
Source
wos-lite
Published In
PloS one
Volume
8
Issue
11
Publish Date
2013
DOI
10.1371/journal.pone.0080532

β-arrestin regulation of myosin light chain phosphorylation promotes AT1aR-mediated cell contraction and migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile 8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility. © 2013 Simard et al.

Authors
Simard, E; Kovacs, JJ; Miller, WE; Kim, J; Grandbois, M; Lefkowitz, RJ
MLA Citation
Simard, E, Kovacs, JJ, Miller, WE, Kim, J, Grandbois, M, and Lefkowitz, RJ. "β-arrestin regulation of myosin light chain phosphorylation promotes AT1aR-mediated cell contraction and migration." PLoS ONE 8.11 (November 8, 2013).
Source
scopus
Published In
PloS one
Volume
8
Issue
11
Publish Date
2013
DOI
10.1371/journal.pone.0080532

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

Authors
Aristotelous, T; Ahn, S; Shukla, AK; Gawron, S; Sassano, MF; Kahsai, AW; Wingler, LM; Zhu, X; Tripathi-Shukla, P; Huang, X-P; Riley, J; Besnard, J; Read, KD; Roth, BL; Gilbert, IH; Hopkins, AL; Lefkowitz, RJ; Navratilova, I
MLA Citation
Aristotelous, T, Ahn, S, Shukla, AK, Gawron, S, Sassano, MF, Kahsai, AW, Wingler, LM, Zhu, X, Tripathi-Shukla, P, Huang, X-P, Riley, J, Besnard, J, Read, KD, Roth, BL, Gilbert, IH, Hopkins, AL, Lefkowitz, RJ, and Navratilova, I. "Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor." ACS medicinal chemistry letters 4.10 (October 2013): 1005-1010.
PMID
24454993
Source
epmc
Published In
ACS Medicinal Chemistry Letters
Volume
4
Issue
10
Publish Date
2013
Start Page
1005
End Page
1010
DOI
10.1021/ml400312j

A brief history of G-protein coupled receptors (Nobel Lecture).

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "A brief history of G-protein coupled receptors (Nobel Lecture)." Angew Chem Int Ed Engl 52.25 (June 17, 2013): 6366-6378.
PMID
23650015
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
52
Issue
25
Publish Date
2013
Start Page
6366
End Page
6378
DOI
10.1002/anie.201301924

Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Authors
Shukla, AK; Manglik, A; Kruse, AC; Xiao, K; Reis, RI; Tseng, W-C; Staus, DP; Hilger, D; Uysal, S; Huang, L-Y; Paduch, M; Tripathi-Shukla, P; Koide, A; Koide, S; Weis, WI; Kossiakoff, AA; Kobilka, BK; Lefkowitz, RJ
MLA Citation
Shukla, AK, Manglik, A, Kruse, AC, Xiao, K, Reis, RI, Tseng, W-C, Staus, DP, Hilger, D, Uysal, S, Huang, L-Y, Paduch, M, Tripathi-Shukla, P, Koide, A, Koide, S, Weis, WI, Kossiakoff, AA, Kobilka, BK, and Lefkowitz, RJ. "Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide." Nature 497.7447 (May 2, 2013): 137-141.
PMID
23604254
Source
pubmed
Published In
Nature
Volume
497
Issue
7447
Publish Date
2013
Start Page
137
End Page
141
DOI
10.1038/nature12120

Crystal structure of active Beta-arrestin1 bound to phosphorylated carboxy-terminus of a G protein-coupled receptor

Authors
Shukla, AK; Manglik, A; Kruse, A; Reis, R; Tseng, WC; Staus, DP; Hilger, D; Uysal, S; Huang, L; Peduch, M; Tripathi-Shukla, P; Koide, A; Koide, S; Weis, WI; Kossiakoff, AK; Kobilka, BK; Lefkowitz, RJ
MLA Citation
Shukla, AK, Manglik, A, Kruse, A, Reis, R, Tseng, WC, Staus, DP, Hilger, D, Uysal, S, Huang, L, Peduch, M, Tripathi-Shukla, P, Koide, A, Koide, S, Weis, WI, Kossiakoff, AK, Kobilka, BK, and Lefkowitz, RJ. "Crystal structure of active Beta-arrestin1 bound to phosphorylated carboxy-terminus of a G protein-coupled receptor." April 2013.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
27
Publish Date
2013

Pharmacological blockade of a β(2)AR-β-arrestin-1 signaling cascade prevents the accumulation of DNA damage in a behavioral stress model.

Chronic stress is known to have a profound negative impact on human health and has been suggested to influence a number of disease states. However, the mechanisms underlying the deleterious effects of stress remain largely unknown. Stress is known to promote the release of epinephrine, a catecholamine stress hormone that binds to β(2)-adrenergic receptors (β(2)ARs) with high affinity. Our previous work has demonstrated that chronic stimulation of a β(2)AR-β-arrestin-1-mediated signaling pathway by infusion of isoproterenol suppresses p53 levels and impairs genomic integrity. In this pathway, β-arrestin-1, which is activated via β(2)ARs, facilitates the AKT-mediated activation of Mdm2 and functions as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, Mdm2. Here, we show that chronic restraint stress in mice recapitulates the effects of isoproterenol infusion to reduce p53 levels and results in the accumulation of DNA damage in the frontal cortex of the brain, two effects that are abrogated by the β-blocker, propranolol and by genetic deletion of β-arrestin-1. These data suggest that the β(2)AR-β-arrestin-1 signaling pathway may represent an attractive therapeutic target to prevent some of the negative consequences of stress in the treatment of stress-related disorders.

Authors
Hara, MR; Sachs, BD; Caron, MG; Lefkowitz, RJ
MLA Citation
Hara, MR, Sachs, BD, Caron, MG, and Lefkowitz, RJ. "Pharmacological blockade of a β(2)AR-β-arrestin-1 signaling cascade prevents the accumulation of DNA damage in a behavioral stress model." Cell Cycle 12.2 (January 15, 2013): 219-224.
PMID
23287463
Source
pubmed
Published In
Cell Cycle
Volume
12
Issue
2
Publish Date
2013
Start Page
219
End Page
224
DOI
10.4161/cc.23368

Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency. © 2013 American Chemical Society.

Authors
Weiss, DR; Ahn, S; Sassano, MF; Kleist, A; Zhu, X; Strachan, R; Roth, BL; Lefkowitz, RJ; Shoichet, BK
MLA Citation
Weiss, DR, Ahn, S, Sassano, MF, Kleist, A, Zhu, X, Strachan, R, Roth, BL, Lefkowitz, RJ, and Shoichet, BK. "Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor." ACS Chemical Biology 8.5 (2013): 1018-1026.
PMID
23485065
Source
scival
Published In
ACS Chemical Biology
Volume
8
Issue
5
Publish Date
2013
Start Page
1018
End Page
1026
DOI
10.1021/cb400103f

Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino-and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins. © 2013 Macmillan Publishers Limited. All rights reserved.

Authors
Shukla, AK; Manglik, A; Kruse, AC; Xiao, K; Reis, RI; Tseng, W-C; Staus, DP; Hilger, D; Uysal, S; Huang, L-Y; Paduch, M; Tripathi-Shukla, P; Koide, A; Koide, S; Weis, WI; Kossiakoff, AA; Kobilka, BK; Lefkowitz, RJ
MLA Citation
Shukla, AK, Manglik, A, Kruse, AC, Xiao, K, Reis, RI, Tseng, W-C, Staus, DP, Hilger, D, Uysal, S, Huang, L-Y, Paduch, M, Tripathi-Shukla, P, Koide, A, Koide, S, Weis, WI, Kossiakoff, AA, Kobilka, BK, and Lefkowitz, RJ. "Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide." Nature 497.7447 (2013): 137-141.
Source
scival
Published In
Nature
Volume
497
Issue
7447
Publish Date
2013
Start Page
137
End Page
141
DOI
10.1038/nature12120

Arrestins come of age: A personal historical perspective

Visual arrestin and the two β-arrestins (1 and 2) were originally discovered 25-30 years ago in the context of their ability to desensitize phosphorylated G protein-coupled receptors (rhodopsin and the β2-adrenergic receptor, respectively). A fourth retinal-specific member of the family (X-arrestin) was discovered later. Over the past 10-15 years, however, it has become clear that these versatile molecules subserve a host of other roles in modulating and mediating the function of most GPCRs as well as other types of receptors. Functioning as multifunctional adaptor proteins, the β-arrestins also play prominent roles in receptor endocytosis, signaling, trafficking, and ubiquitination among others. Here, I provide a brief personal perspective on how the field has evolved since its inception and speculate on future directions. © 2013 Elsevier Inc.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Arrestins come of age: A personal historical perspective." Progress in Molecular Biology and Translational Science 118 (2013): 3-18.
PMID
23764048
Source
scival
Published In
Progress in Molecular Biology and Translational Science
Volume
118
Publish Date
2013
Start Page
3
End Page
18
DOI
10.1016/B978-0-12-394440-5.00001-2

β-Arrestin2 mediates the initiation and progression of myeloid leukemia.

β-Arrestins were initially discovered as negative regulators of G protein-coupled receptor signaling. Although β-arrestins have more recently been implicated as scaffold proteins that interact with various mitogenic and developmental signals, the genetic role of β-arrestins in driving oncogenesis is not known. Here we have investigated the role of β-arrestin in hematologic malignancies and have found that although both β-arrestin1 and -2 are expressed in the hematopoietic system, loss of β-arrestin2 preferentially leads to a severe impairment in the establishment and propagation of the chronic and blast crisis phases of chronic myelogenous leukemia (CML). These defects are linked to a reduced frequency, as well as defective self-renewal capacity of the cancer stem-cell population, in mouse models and in human CML patient samples. At a molecular level, the loss of β-arrestin2 leads to a significant inhibition of β-catenin stabilization, and ectopic activation of Wnt signaling reverses the defects observed in the β-arrestin2 mutant cells. These data cumulatively show that β-arrestin2 is essential for CML disease propagation and indicate that β-arrestins and the Wnt/β-catenin pathway lie in a signaling hierarchy in the context of CML cancer stem cell maintenance.

Authors
Fereshteh, M; Ito, T; Kovacs, JJ; Zhao, C; Kwon, HY; Tornini, V; Konuma, T; Chen, M; Lefkowitz, RJ; Reya, T
MLA Citation
Fereshteh, M, Ito, T, Kovacs, JJ, Zhao, C, Kwon, HY, Tornini, V, Konuma, T, Chen, M, Lefkowitz, RJ, and Reya, T. "β-Arrestin2 mediates the initiation and progression of myeloid leukemia." Proc Natl Acad Sci U S A 109.31 (July 31, 2012): 12532-12537.
PMID
22773819
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
31
Publish Date
2012
Start Page
12532
End Page
12537
DOI
10.1073/pnas.1209815109

Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

Authors
Heitzler, D; Durand, G; Gallay, N; Rizk, A; Ahn, S; Kim, J; Violin, JD; Dupuy, L; Gauthier, C; Piketty, V; Crépieux, P; Poupon, A; Clément, F; Fages, F; Lefkowitz, RJ; Reiter, E
MLA Citation
Heitzler, D, Durand, G, Gallay, N, Rizk, A, Ahn, S, Kim, J, Violin, JD, Dupuy, L, Gauthier, C, Piketty, V, Crépieux, P, Poupon, A, Clément, F, Fages, F, Lefkowitz, RJ, and Reiter, E. "Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling. (Published online)" Mol Syst Biol 8 (June 26, 2012): 590-.
PMID
22735336
Source
pubmed
Published In
Molecular systems biology
Volume
8
Publish Date
2012
Start Page
590
DOI
10.1038/msb.2012.22

Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors.

The concept of biased agonism has recently come to the fore with the realization that seven-transmembrane receptors (7TMRs, also known as G protein-coupled receptors, or GPCRs) activate complex signaling networks and can adopt multiple active conformations upon agonist binding. As a consequence, the "efficacy" of receptors, which was classically considered linear, is now recognized as pluridimensional. Biased agonists selectively stabilize only a subset of receptor conformations induced by the natural "unbiased" ligand, thus preferentially activating certain signaling mechanisms. Such agonists thus reveal the intriguing possibility that one can direct cellular signaling with unprecedented precision and specificity and support the notion that biased agonists may identify new classes of therapeutic agents that have fewer side effects. This review focuses on one particular class of biased ligands that has the ability to alter the balance between G protein-dependent and β-arrestin-dependent signal transduction.

Authors
Reiter, E; Ahn, S; Shukla, AK; Lefkowitz, RJ
MLA Citation
Reiter, E, Ahn, S, Shukla, AK, and Lefkowitz, RJ. "Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors." Annu Rev Pharmacol Toxicol 52 (2012): 179-197. (Review)
PMID
21942629
Source
pubmed
Published In
Annual Review of Pharmacology and Toxicology
Volume
52
Publish Date
2012
Start Page
179
End Page
197
DOI
10.1146/annurev.pharmtox.010909.105800

Science Signaling Podcast: 20 November 2012

This Podcast features an interview with Robert Lefkowitz, corecipient of this year's Nobel Prize in Chemistry. Lefkowitz has been studying the function of G protein-coupled receptors (GPCRs) for 40 years, and his laboratory has made many important contributions to our current understanding of GPCR structure and function. GPCRs are integral membrane proteins with seven transmembrane- spanning domains. The extracellular portions of the receptors bind to ligands outside the cell, and the intracellular portions of these receptors transduce signaling. GPCRs control many different aspects of cell biology and participate in the regulation of many physiological processes. A large percentage of drugs in use today target GPCRs or their associated signal transduction partners. Lefkowitz discusses GPCR function and specificity, his role in this field of research, and future challenges.

Authors
Lefkowitz, RJ; VanHook, AM
MLA Citation
Lefkowitz, RJ, and VanHook, AM. "Science Signaling Podcast: 20 November 2012." Science Signaling 5.251 (2012).
Source
scival
Published In
Science Signaling
Volume
5
Issue
251
Publish Date
2012
DOI
10.1126/scisignal.2003756

Global phosphorylation analysis of β-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR) (Proceedings of the National Academy of Sciences of the United States of America (2010) 107, 34 (15299-15304) doi:10.1073/pnas.1008461107)

Authors
Xiao, K; Sun, J; Kim, J; Rajagopal, S; Zhai, B; Villén, J; Haas, W; Kovacs, JJ; Shukla, AK; Hara, MR; Hernandez, M; Lachmann, A; Zhao, S; Lin, Y; Cheng, Y; Mizuno, K; Ma'ayan, A; Gygi, SP; Lefkowitz, RJ
MLA Citation
Xiao, K, Sun, J, Kim, J, Rajagopal, S, Zhai, B, Villén, J, Haas, W, Kovacs, JJ, Shukla, AK, Hara, MR, Hernandez, M, Lachmann, A, Zhao, S, Lin, Y, Cheng, Y, Mizuno, K, Ma'ayan, A, Gygi, SP, and Lefkowitz, RJ. "Global phosphorylation analysis of β-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR) (Proceedings of the National Academy of Sciences of the United States of America (2010) 107, 34 (15299-15304) doi:10.1073/pnas.1008461107)." Proceedings of the National Academy of Sciences of the United States of America 109.33 (2012): 13464--.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
33
Publish Date
2012
Start Page
13464-
DOI
10.1073/pnas.1211889109

An interview with Robert J. Lefkowitz.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "An interview with Robert J. Lefkowitz." Trends in pharmacological sciences 33.2 (2012): 51-52.
PMID
22403833
Source
scival
Published In
Trends in Pharmacological Sciences
Volume
33
Issue
2
Publish Date
2012
Start Page
51
End Page
52

A signal honour

Authors
Lefkowitz, R; Kobilka, B
MLA Citation
Lefkowitz, R, and Kobilka, B. "A signal honour." Chemistry World 9.11 (2012): 46-49.
Source
scival
Published In
Chemistry World
Volume
9
Issue
11
Publish Date
2012
Start Page
46
End Page
49

A tale of two callings.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "A tale of two callings." J Clin Invest 121.10 (October 2011): 4201-4203.
Website
http://hdl.handle.net/10161/7788
PMID
21965340
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
121
Issue
10
Publish Date
2011
Start Page
4201
End Page
4203
DOI
10.1172/JCI60817

β-Arrestin-mediated receptor trafficking and signal transduction.

β-Arrestins function as endocytic adaptors and mediate trafficking of a variety of cell-surface receptors, including seven-transmembrane receptors (7TMRs). In the case of 7TMRs, β-arrestins carry out these tasks while simultaneously inhibiting upstream G-protein-dependent signaling and promoting alternate downstream signaling pathways. The mechanisms by which β-arrestins interact with a continuously expanding ensemble of protein partners and perform their multiple functions including trafficking and signaling are currently being uncovered. Molecular changes at the level of protein conformation as well as post-translational modifications of β-arrestins probably form the basis for their dynamic interactions during receptor trafficking and signaling. It is becoming increasingly evident that β-arrestins, originally discovered as 7TMR adaptor proteins, indeed have much broader and more versatile roles in maintaining cellular homeostasis. In this review paper, we assess the traditional and novel functions of β-arrestins and discuss the molecular attributes that might facilitate multiple interactions in regulating cell signaling and receptor trafficking.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "β-Arrestin-mediated receptor trafficking and signal transduction." Trends Pharmacol Sci 32.9 (September 2011): 521-533. (Review)
PMID
21680031
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
32
Issue
9
Publish Date
2011
Start Page
521
End Page
533
DOI
10.1016/j.tips.2011.05.002

Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling.

β-Arrestins, originally discovered to desensitize activated seven transmembrane receptors (7TMRs; also known as G-protein-coupled receptors, GPCRs), are now well established mediators of receptor endocytosis, ubiquitylation and G protein-independent signaling. Recent global analyses of β-arrestin interactions and β-arrestin-dependent phosphorylation events have uncovered several previously unanticipated roles of β-arrestins in a range of cellular signaling events. These findings strongly suggest that the functional roles of β-arrestins are much broader than currently understood. Biophysical studies aimed at understanding multiple active conformations of the 7TMRs and the β-arrestins have begun to unravel the mechanistic basis for the diverse functional capabilities of β-arrestins in cellular signaling.

Authors
Shukla, AK; Xiao, K; Lefkowitz, RJ
MLA Citation
Shukla, AK, Xiao, K, and Lefkowitz, RJ. "Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling." Trends Biochem Sci 36.9 (September 2011): 457-469. (Review)
PMID
21764321
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
36
Issue
9
Publish Date
2011
Start Page
457
End Page
469
DOI
10.1016/j.tibs.2011.06.003

Quantifying ligand bias at seven-transmembrane receptors.

Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, β-arrestins. Biased agonists, which signal with different efficacies to a receptor's multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the β2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.

Authors
Rajagopal, S; Ahn, S; Rominger, DH; Gowen-MacDonald, W; Lam, CM; Dewire, SM; Violin, JD; Lefkowitz, RJ
MLA Citation
Rajagopal, S, Ahn, S, Rominger, DH, Gowen-MacDonald, W, Lam, CM, Dewire, SM, Violin, JD, and Lefkowitz, RJ. "Quantifying ligand bias at seven-transmembrane receptors." Molecular pharmacology 80.3 (September 2011): 367-377.
PMID
21610196
Source
epmc
Published In
Molecular pharmacology
Volume
80
Issue
3
Publish Date
2011
Start Page
367
End Page
377
DOI
10.1124/mol.111.072801

Multiple ligand-specific conformations of the β2-adrenergic receptor.

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

Authors
Kahsai, AW; Xiao, K; Rajagopal, S; Ahn, S; Shukla, AK; Sun, J; Oas, TG; Lefkowitz, RJ
MLA Citation
Kahsai, AW, Xiao, K, Rajagopal, S, Ahn, S, Shukla, AK, Sun, J, Oas, TG, and Lefkowitz, RJ. "Multiple ligand-specific conformations of the β2-adrenergic receptor. (Published online)" Nat Chem Biol 7.10 (August 21, 2011): 692-700.
PMID
21857662
Source
pubmed
Published In
Nature Chemical Biology
Volume
7
Issue
10
Publish Date
2011
Start Page
692
End Page
700
DOI
10.1038/nchembio.634

A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1.

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

Authors
Hara, MR; Kovacs, JJ; Whalen, EJ; Rajagopal, S; Strachan, RT; Grant, W; Towers, AJ; Williams, B; Lam, CM; Xiao, K; Shenoy, SK; Gregory, SG; Ahn, S; Duckett, DR; Lefkowitz, RJ
MLA Citation
Hara, MR, Kovacs, JJ, Whalen, EJ, Rajagopal, S, Strachan, RT, Grant, W, Towers, AJ, Williams, B, Lam, CM, Xiao, K, Shenoy, SK, Gregory, SG, Ahn, S, Duckett, DR, and Lefkowitz, RJ. "A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1. (Published online)" Nature 477.7364 (August 21, 2011): 349-353.
PMID
21857681
Source
pubmed
Published In
Nature
Volume
477
Issue
7364
Publish Date
2011
Start Page
349
End Page
353
DOI
10.1038/nature10368

Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.

Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β(2)-adrenergic receptor (β(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and β-arrestin-mediated pathways or with a biased ligand that promotes signaling only through β-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Authors
Nobles, KN; Xiao, K; Ahn, S; Shukla, AK; Lam, CM; Rajagopal, S; Strachan, RT; Huang, T-Y; Bressler, EA; Hara, MR; Shenoy, SK; Gygi, SP; Lefkowitz, RJ
MLA Citation
Nobles, KN, Xiao, K, Ahn, S, Shukla, AK, Lam, CM, Rajagopal, S, Strachan, RT, Huang, T-Y, Bressler, EA, Hara, MR, Shenoy, SK, Gygi, SP, and Lefkowitz, RJ. "Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin." Sci Signal 4.185 (August 9, 2011): ra51-.
PMID
21868357
Source
pubmed
Published In
Science Signaling
Volume
4
Issue
185
Publish Date
2011
Start Page
ra51
DOI
10.1126/scisignal.2001707

Introduction to the series on novel aspects of cardiovascular G-protein-coupled receptor signaling.

Authors
Rockman, HA; Lefkowitz, RJ
MLA Citation
Rockman, HA, and Lefkowitz, RJ. "Introduction to the series on novel aspects of cardiovascular G-protein-coupled receptor signaling." Circ Res 109.2 (July 8, 2011): 202-204.
PMID
21737815
Source
pubmed
Published In
Circulation Research
Volume
109
Issue
2
Publish Date
2011
Start Page
202
End Page
204
DOI
10.1161/CIRCRESAHA.110.231126

BETA-ARRESTINS REGULATE SIGNALING BY BONE MORPHOGENETIC PROTEIN TYPE II RECEPTOR IN PULMONARY ARTERIAL HYPERTENSION

Authors
Rajagopal, S; Kovacs, J; Badea, C; Johnson, GA; Rockman, HA; Piantadosi, CA; Lefkowitz, RJ
MLA Citation
Rajagopal, S, Kovacs, J, Badea, C, Johnson, GA, Rockman, HA, Piantadosi, CA, and Lefkowitz, RJ. "BETA-ARRESTINS REGULATE SIGNALING BY BONE MORPHOGENETIC PROTEIN TYPE II RECEPTOR IN PULMONARY ARTERIAL HYPERTENSION." JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 57.14 (April 5, 2011): E2046-E2046.
Source
wos-lite
Published In
JACC - Journal of the American College of Cardiology
Volume
57
Issue
14
Publish Date
2011
Start Page
E2046
End Page
E2046

β-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix.

Idiopathic pulmonary fibrosis is a progressive disease that causes unremitting extracellular matrix deposition with resulting distortion of pulmonary architecture and impaired gas exchange. β-Arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Here, we examine the role of β-arrestin1 and β-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of either β-arrestin1 or β-arrestin2 resulted in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis was prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated β-arrestin-null mice failed to invade extracellular matrix and displayed altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of β-arrestin2 in fibroblasts from patients with idiopathic pulmonary fibrosis attenuated the invasive phenotype. These data implicate β-arrestins as mediators of fibroblast invasion and the development of pulmonary fibrosis, and as a potential target for therapeutic intervention in patients with idiopathic pulmonary fibrosis.

Authors
Lovgren, AK; Kovacs, JJ; Xie, T; Potts, EN; Li, Y; Foster, WM; Liang, J; Meltzer, EB; Jiang, D; Lefkowitz, RJ; Noble, PW
MLA Citation
Lovgren, AK, Kovacs, JJ, Xie, T, Potts, EN, Li, Y, Foster, WM, Liang, J, Meltzer, EB, Jiang, D, Lefkowitz, RJ, and Noble, PW. "β-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix." Sci Transl Med 3.74 (March 16, 2011): 74ra23-.
PMID
21411739
Source
pubmed
Published In
Science Translational Medicine
Volume
3
Issue
74
Publish Date
2011
Start Page
74ra23
DOI
10.1126/scitranslmed.3001564

Therapeutic potential of β-arrestin- and G protein-biased agonists.

Members of the seven-transmembrane receptor (7TMR), or G protein-coupled receptor (GPCR), superfamily represent some of the most successful targets of modern drug therapy, with proven efficacy in the treatment of a broad range of human conditions and disease processes. It is now appreciated that β-arrestins, once viewed simply as negative regulators of traditional 7TMR-stimulated G protein signaling, act as multifunctional adapter proteins that regulate 7TMR desensitization and trafficking and promote distinct intracellular signals in their own right. Moreover, several 7TMR biased agonists, which selectively activate these divergent signaling pathways, have been identified. Here we highlight the diversity of G protein- and β-arrestin-mediated functions and the therapeutic potential of selective targeting of these in disease states.

Authors
Whalen, EJ; Rajagopal, S; Lefkowitz, RJ
MLA Citation
Whalen, EJ, Rajagopal, S, and Lefkowitz, RJ. "Therapeutic potential of β-arrestin- and G protein-biased agonists." Trends in molecular medicine 17.3 (March 2011): 126-139. (Review)
PMID
21183406
Source
epmc
Published In
Trends in Molecular Medicine
Volume
17
Issue
3
Publish Date
2011
Start Page
126
End Page
139
DOI
10.1016/j.molmed.2010.11.004

Arresting a transient receptor potential (TRP) channel: beta-arrestin 1 mediates ubiquitination and functional down-regulation of TRPV4.

β-Arrestins, originally discovered to desensitize activated G protein-coupled receptors, (aka seven-transmembrane receptors, 7TMRs) also mediate 7TMR internalization and G protein-independent signaling via these receptors. More recently, several regulatory roles of β-arrestins for atypical 7TMRs and non-7TM receptors have emerged. Here, we uncover an entirely novel regulatory role of β-arrestins in cross-talk between the angiotensin receptor (AT1aR) and a member of the transient receptor potential (TRP) ion channel family, TRPV4. AT1aR and TRPV4 form a constitutive complex in the plasma membrane, and angiotensin stimulation leads to recruitment of β-arrestin 1 to this complex. Surprisingly, angiotensin stimulation results in ubiquitination of TRPV4, a process that requires β-arrestin 1, and subsequently to internalization and functional down-regulation of TRPV4. β-Arrestin 1 interacts with, and acts as an adaptor for AIP4, an E3 ubiquitin ligase responsible for TRPV4 ubiquitination. Thus, our data provide the first evidence of a functional link between β-arrestins and TRPV4 and uncovers an entirely novel mechanism to maintain appropriate intracellular Ca(2+) concentration to avoid excessive Ca(2+) signaling.

Authors
Shukla, AK; Kim, J; Ahn, S; Xiao, K; Shenoy, SK; Liedtke, W; Lefkowitz, RJ
MLA Citation
Shukla, AK, Kim, J, Ahn, S, Xiao, K, Shenoy, SK, Liedtke, W, and Lefkowitz, RJ. "Arresting a transient receptor potential (TRP) channel: beta-arrestin 1 mediates ubiquitination and functional down-regulation of TRPV4." J Biol Chem 285.39 (September 24, 2010): 30115-30125.
PMID
20650893
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
39
Publish Date
2010
Start Page
30115
End Page
30125
DOI
10.1074/jbc.M110.141549

Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).

beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.

Authors
Xiao, K; Sun, J; Kim, J; Rajagopal, S; Zhai, B; Villén, J; Haas, W; Kovacs, JJ; Shukla, AK; Hara, MR; Hernandez, M; Lachmann, A; Zhao, S; Lin, Y; Cheng, Y; Mizuno, K; Ma'ayan, A; Gygi, SP; Lefkowitz, RJ
MLA Citation
Xiao, K, Sun, J, Kim, J, Rajagopal, S, Zhai, B, Villén, J, Haas, W, Kovacs, JJ, Shukla, AK, Hara, MR, Hernandez, M, Lachmann, A, Zhao, S, Lin, Y, Cheng, Y, Mizuno, K, Ma'ayan, A, Gygi, SP, and Lefkowitz, RJ. "Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR)." Proc Natl Acad Sci U S A 107.34 (August 24, 2010): 15299-15304.
PMID
20686112
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
34
Publish Date
2010
Start Page
15299
End Page
15304
DOI
10.1073/pnas.1008461107

The Unique Efficacy Profile of a beta-Arrestin Pathway-Selective Parathyroid Hormone Receptor Agonist Revealed by Metabolic Pathways Analysis.

Authors
Maudsley, S; Liao, S; Yuan, L; Lefkowitz, RJ; Luttrell, LM; Gesty-Palmer, D
MLA Citation
Maudsley, S, Liao, S, Yuan, L, Lefkowitz, RJ, Luttrell, LM, and Gesty-Palmer, D. "The Unique Efficacy Profile of a beta-Arrestin Pathway-Selective Parathyroid Hormone Receptor Agonist Revealed by Metabolic Pathways Analysis." June 2010.
Source
wos-lite
Published In
Endocrine reviews
Volume
31
Issue
3
Publish Date
2010

Teaching old receptors new tricks: biasing seven-transmembrane receptors.

Seven-transmembrane receptors (7TMRs; also known as G protein-coupled receptors) are the largest class of receptors in the human genome and are common targets for therapeutics. Originally identified as mediators of 7TMR desensitization, beta-arrestins (arrestin 2 and arrestin 3) are now recognized as true adaptor proteins that transduce signals to multiple effector pathways. Signalling that is mediated by beta-arrestins has distinct biochemical and functional consequences from those mediated by G proteins, and several biased ligands and receptors have been identified that preferentially signal through either G protein- or beta-arrestin-mediated pathways. These ligands are not only useful tools for investigating the biochemistry of 7TMR signalling, they also have the potential to be developed into new classes of therapeutics.

Authors
Rajagopal, S; Rajagopal, K; Lefkowitz, RJ
MLA Citation
Rajagopal, S, Rajagopal, K, and Lefkowitz, RJ. "Teaching old receptors new tricks: biasing seven-transmembrane receptors." Nature reviews. Drug discovery 9.5 (May 2010): 373-386. (Review)
PMID
20431569
Source
epmc
Published In
Nature Reviews Drug Discovery
Volume
9
Issue
5
Publish Date
2010
Start Page
373
End Page
386
DOI
10.1038/nrd3024

Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7.

Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through beta-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both beta-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling "decoys" because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through beta-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or beta-arrestin depletion by siRNA. This example of an endogenous "beta-arrestin-biased" 7TMR that signals through beta-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through beta-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.

Authors
Rajagopal, S; Kim, J; Ahn, S; Craig, S; Lam, CM; Gerard, NP; Gerard, C; Lefkowitz, RJ
MLA Citation
Rajagopal, S, Kim, J, Ahn, S, Craig, S, Lam, CM, Gerard, NP, Gerard, C, and Lefkowitz, RJ. "Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7." Proceedings of the National Academy of Sciences of the United States of America 107.2 (January 2010): 628-632.
PMID
20018651
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
2
Publish Date
2010
Start Page
628
End Page
632
DOI
10.1073/pnas.0912852107

International workshop at the nobel forum, karolinska institutet on g protein-coupled receptors: Finding the words to describe monomers, oligomers, and their molecular mechanisms and defining their meaning. Can a consensus be reached

A meeting was held May 19, 2010 at the Karolinski Institute on Nomenclature in Pharmacology. This meeting occurred in conjunction with the Symposium The Changing World of G Protein Coupled Receptors: From Monomers to Dimers and Receptor Mosaics (Higher-order Oligomers) held the previous day at the Royal Swedish Academy of Science. Two broad topics of nomenclature were discussed; ligand nomenclature and the definition of 'receptorreceptor' interactions. This paper summarizes discussions on these topics along with a consensus definition of the term 'receptorreceptor' interaction. © 2010 Informa Healthcare USA, Inc.

Authors
Kenakin, T; Agnati, LF; Caron, M; Fredholm, B; Guidoli, D; Kobilka, B; Lefkowitz, RW; Lohse, M; Woods, A; Fuxe, K
MLA Citation
Kenakin, T, Agnati, LF, Caron, M, Fredholm, B, Guidoli, D, Kobilka, B, Lefkowitz, RW, Lohse, M, Woods, A, and Fuxe, K. "International workshop at the nobel forum, karolinska institutet on g protein-coupled receptors: Finding the words to describe monomers, oligomers, and their molecular mechanisms and defining their meaning. Can a consensus be reached." Journal of Receptors and Signal Transduction 30.5 (2010): 284-286.
PMID
20858022
Source
scival
Published In
Journal of Receptors and Signal Transduction (Informa)
Volume
30
Issue
5
Publish Date
2010
Start Page
284
End Page
286
DOI
10.3109/10799893.2010.512438

G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3beta, a cytosolic kinase for phosphorylation of a downstream effector beta-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of beta-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished beta-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Authors
Chen, M; Philipp, M; Wang, J; Premont, RT; Garrison, TR; Caron, MG; Lefkowitz, RJ; Chen, W
MLA Citation
Chen, M, Philipp, M, Wang, J, Premont, RT, Garrison, TR, Caron, MG, Lefkowitz, RJ, and Chen, W. "G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway." J Biol Chem 284.50 (December 11, 2009): 35040-35048.
PMID
19801552
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
50
Publish Date
2009
Start Page
35040
End Page
35048
DOI
10.1074/jbc.M109.047456

A Modified beta 1-adrenergic Receptor Demonstrates Bias Towards G Protein Receptor Kinase Phosphorylation

Authors
Patel, CB; Patel, PA; Frangakis, SG; Chen, M; Shenoy, SK; Lefkowitz, RJ; Rockman, HA
MLA Citation
Patel, CB, Patel, PA, Frangakis, SG, Chen, M, Shenoy, SK, Lefkowitz, RJ, and Rockman, HA. "A Modified beta 1-adrenergic Receptor Demonstrates Bias Towards G Protein Receptor Kinase Phosphorylation." November 3, 2009.
Source
wos-lite
Published In
Circulation
Volume
120
Issue
18
Publish Date
2009
Start Page
S800
End Page
S801

Science Translational Medicine Podcast: 7 October 2009

Authors
Gesty-Palmer, D; Nabel, B; Lefkowitz, RJ; Kelner, KL
MLA Citation
Gesty-Palmer, D, Nabel, B, Lefkowitz, RJ, and Kelner, KL. "Science Translational Medicine Podcast: 7 October 2009." Science Translational Medicine 1.1 (October 7, 2009): 1pc1-1pc1.
Source
crossref
Published In
Science Translational Medicine
Volume
1
Issue
1
Publish Date
2009
Start Page
1pc1
End Page
1pc1
DOI
10.1126/scitranslmed.1pc1

A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation.

About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through beta-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp(12),Tyr(34))-PTH(7-34) (PTH-betaarr), which activates beta-arrestin but not classic G protein signaling. In mice, PTH-betaarr induces anabolic bone formation, as does the nonselective agonist PTH(1-34), which activates both mechanisms. In beta-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-betaarr is ablated. The beta-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the beta-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.

Authors
Gesty-Palmer, D; Flannery, P; Yuan, L; Corsino, L; Spurney, R; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Gesty-Palmer, D, Flannery, P, Yuan, L, Corsino, L, Spurney, R, Lefkowitz, RJ, and Luttrell, LM. "A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation." Sci Transl Med 1.1 (October 7, 2009): 1ra1-.
PMID
20368153
Source
pubmed
Published In
Science Translational Medicine
Volume
1
Issue
1
Publish Date
2009
Start Page
1ra1
DOI
10.1126/scitranslmed.3000071

Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways.

Arrestins were identified as mediators of G protein-coupled receptor (GPCR) desensitization and endocytosis. However, it is now clear that they scaffold many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors--including those relevant to the Hedgehog, Wnt, Notch, and TGFbeta pathways--and downstream kinases such as the MAPK and Akt/PI3K cascades. The involvement of arrestins in many discrete developmental signaling events suggests an indispensable role for these multifaceted molecular scaffolds.

Authors
Kovacs, JJ; Hara, MR; Davenport, CL; Kim, J; Lefkowitz, RJ
MLA Citation
Kovacs, JJ, Hara, MR, Davenport, CL, Kim, J, and Lefkowitz, RJ. "Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways." Dev Cell 17.4 (October 2009): 443-458. (Review)
PMID
19853559
Source
pubmed
Published In
Developmental Cell
Volume
17
Issue
4
Publish Date
2009
Start Page
443
End Page
458
DOI
10.1016/j.devcel.2009.09.011

Oxygen-regulated beta(2)-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL.

Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although beta-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the beta(2)-adrenergic receptor (beta(2)AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the beta(2)AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with beta(2)AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the beta(2)AR to serve as an endogenous beta(2)AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous beta(2)AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.

Authors
Xie, L; Xiao, K; Whalen, EJ; Forrester, MT; Freeman, RS; Fong, G; Gygi, SP; Lefkowitz, RJ; Stamler, JS
MLA Citation
Xie, L, Xiao, K, Whalen, EJ, Forrester, MT, Freeman, RS, Fong, G, Gygi, SP, Lefkowitz, RJ, and Stamler, JS. "Oxygen-regulated beta(2)-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL. (Published online)" Sci Signal 2.78 (July 7, 2009): ra33-.
PMID
19584355
Source
pubmed
Published In
Science Signaling
Volume
2
Issue
78
Publish Date
2009
Start Page
ra33
DOI
10.1126/scisignal.2000444

Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands.

CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ss-arrestin system. CCL19 leads to robust CCR7 phosphorylation and beta-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to beta-arrestin2 recruitment, only CCL19 leads to redistribution of beta-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and beta-arrestin2 to ERK kinases. Thus, this mechanism for "ligand bias" whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of beta-arrestin.

Authors
Zidar, DA; Violin, JD; Whalen, EJ; Lefkowitz, RJ
MLA Citation
Zidar, DA, Violin, JD, Whalen, EJ, and Lefkowitz, RJ. "Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands." Proc Natl Acad Sci U S A 106.24 (June 16, 2009): 9649-9654.
PMID
19497875
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
24
Publish Date
2009
Start Page
9649
End Page
9654
DOI
10.1073/pnas.0904361106

Independent beta-arrestin2 and Gq/protein kinase Czeta pathways for ERK stimulated by angiotensin type 1A receptors in vascular smooth muscle cells converge on transactivation of the epidermal growth factor receptor.

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and beta-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the beta-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated beta-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of beta-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Czeta. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. beta-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and beta-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.

Authors
Kim, J; Ahn, S; Rajagopal, K; Lefkowitz, RJ
MLA Citation
Kim, J, Ahn, S, Rajagopal, K, and Lefkowitz, RJ. "Independent beta-arrestin2 and Gq/protein kinase Czeta pathways for ERK stimulated by angiotensin type 1A receptors in vascular smooth muscle cells converge on transactivation of the epidermal growth factor receptor." J Biol Chem 284.18 (May 1, 2009): 11953-11962.
PMID
19254952
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
18
Publish Date
2009
Start Page
11953
End Page
11962
DOI
10.1074/jbc.M808176200

beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

Authors
Walters, RW; Shukla, AK; Kovacs, JJ; Violin, JD; DeWire, SM; Lam, CM; Chen, JR; Muehlbauer, MJ; Whalen, EJ; Lefkowitz, RJ
MLA Citation
Walters, RW, Shukla, AK, Kovacs, JJ, Violin, JD, DeWire, SM, Lam, CM, Chen, JR, Muehlbauer, MJ, Whalen, EJ, and Lefkowitz, RJ. "beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice." J Clin Invest 119.5 (May 2009): 1312-1321.
Website
http://hdl.handle.net/10161/5928
PMID
19349687
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
119
Issue
5
Publish Date
2009
Start Page
1312
End Page
1321
DOI
10.1172/JCI36806

Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2.

Beta-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seven-transmembrane receptors (7TMRs). Agonist-stimulated ubiquitination of beta-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid beta(2)-adrenergic receptor (beta(2)AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds beta-arrestin2 and leads to the deubiquitination of beta-arrestins. USP33 and Mdm2 function reciprocally and favor respectively the stability or lability of the receptor beta-arrestin complex, thus regulating the longevity and subcellular localization of receptor signalosomes. Receptors such as the beta(2)AR, previously shown to form loose complexes with beta-arrestin ("class A") promote a beta-arrestin conformation conducive for binding to the deubiquitinase, whereas the vasopressin V2R, which forms tight beta-arrestin complexes ("class B"), promotes a distinct beta-arrestin conformation that favors dissociation of the enzyme. Thus, USP33-beta-arrestin interaction is a key regulatory step in 7TMR trafficking and signal transmission from the activated receptors to downstream effectors.

Authors
Shenoy, SK; Modi, AS; Shukla, AK; Xiao, K; Berthouze, M; Ahn, S; Wilkinson, KD; Miller, WE; Lefkowitz, RJ
MLA Citation
Shenoy, SK, Modi, AS, Shukla, AK, Xiao, K, Berthouze, M, Ahn, S, Wilkinson, KD, Miller, WE, and Lefkowitz, RJ. "Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2." Proc Natl Acad Sci U S A 106.16 (April 21, 2009): 6650-6655.
PMID
19363159
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
16
Publish Date
2009
Start Page
6650
End Page
6655
DOI
10.1073/pnas.0901083106

Beta-arrestin1 mediates nicotinic acid induced flushing, but not its antilipolytic effect

Authors
Walters, RW; Shukla, AK; Kovacs, JJ; Violin, JD; DeWire, SM; Lam, CM; Chen, JR; Muehlbauer, MJ; Whalen, EJ; Lefkowitz, RJ
MLA Citation
Walters, RW, Shukla, AK, Kovacs, JJ, Violin, JD, DeWire, SM, Lam, CM, Chen, JR, Muehlbauer, MJ, Whalen, EJ, and Lefkowitz, RJ. "Beta-arrestin1 mediates nicotinic acid induced flushing, but not its antilipolytic effect." JOURNAL OF INVESTIGATIVE DERMATOLOGY 129 (April 2009): S1-S1.
Source
wos-lite
Published In
Journal of Investigative Dermatology
Volume
129
Publish Date
2009
Start Page
S1
End Page
S1

[beta]-arrestin 1 mediates angiotensin II induced ubiquitination and down-regulation of TRPV4

Authors
Shukla, AK; Kim, J; Ahn, S; Xiao, K; Liedtke, W; Lefkowitz, RJ
MLA Citation
Shukla, AK, Kim, J, Ahn, S, Xiao, K, Liedtke, W, and Lefkowitz, RJ. "[beta]-arrestin 1 mediates angiotensin II induced ubiquitination and down-regulation of TRPV4." FASEB JOURNAL 23 (April 2009).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
23
Publish Date
2009

{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.

beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate beta-arrestin but not G protein-dependent signaling, and are abrogated by beta-arrestin-2 small interfering RNA. These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.

Authors
Ahn, S; Kim, J; Hara, MR; Ren, X-R; Lefkowitz, RJ
MLA Citation
Ahn, S, Kim, J, Hara, MR, Ren, X-R, and Lefkowitz, RJ. "{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation." J Biol Chem 284.13 (March 27, 2009): 8855-8865.
PMID
19171933
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
13
Publish Date
2009
Start Page
8855
End Page
8865
DOI
10.1074/jbc.M808463200

Focus on the research journey. An interview with Robert J. Lefkowitz, MD, scientist, mentor, and recipient of the 2007 National Medal of Science by Elaine Musgrave.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Focus on the research journey. An interview with Robert J. Lefkowitz, MD, scientist, mentor, and recipient of the 2007 National Medal of Science by Elaine Musgrave." Clinical and translational science 2.1 (2009): 4-5.
PMID
20443860
Source
scival
Published In
Clinical and Translational Science
Volume
2
Issue
1
Publish Date
2009
Start Page
4
End Page
5
DOI
10.1111/j.1752-8062.2008.00081.x

Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN).

OBJECTIVE: Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model. METHODS: Clinical, histologic, and cytokine responses in GRK6-/-, GRK5-/-, GRK2+/-, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays. RESULTS: Both GRK6-/- and GRK2+/- mice had increased arthritis disease severity (p<0.001); whereas GRK5-/- was not different from controls. Acute weight loss was enhanced in GRK6-/- and GRK2+/- mice (p<0.001, days 3-10). However, GRK6-/- mice uniquely had more weight loss (>10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro. CONCLUSIONS: GRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.

Authors
Tarrant, TK; Rampersad, RR; Esserman, D; Rothlein, LR; Liu, P; Premont, RT; Lefkowitz, RJ; Lee, DM; Patel, DD
MLA Citation
Tarrant, TK, Rampersad, RR, Esserman, D, Rothlein, LR, Liu, P, Premont, RT, Lefkowitz, RJ, Lee, DM, and Patel, DD. "Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN)." Clin Immunol 129.1 (October 2008): 115-122.
PMID
18662895
Source
pubmed
Published In
Clinical Immunology
Volume
129
Issue
1
Publish Date
2008
Start Page
115
End Page
122
DOI
10.1016/j.clim.2008.06.008

Pharmacological characterization of membrane-expressed human trace amine-associated receptor 1 (TAAR1) by a bioluminescence resonance energy transfer cAMP biosensor.

Trace amines are neurotransmitters whose role in regulating invertebrate physiology has been appreciated for many decades. Recent studies indicate that trace amines may also play a role in mammalian physiology by binding to a novel family of G protein-coupled receptors (GPCRs) that are found throughout the central nervous system. A major obstacle impeding the careful pharmacological characterization of trace amine associated receptors (TAARs) is their extremely poor membrane expression in model cell systems, and a molecular basis for this phenomenon has not been determined. In the present study, we show that the addition of an asparagine-linked glycosylation site to the N terminus of the human trace amine associated receptor 1 (TAAR1) is sufficient to enable its plasma membrane expression, and thus its pharmacological characterization with a novel cAMP EPAC (exchange protein directly activated by cAMP) protein based bioluminescence resonance energy transfer (BRET) biosensor. We applied this novel cAMP BRET biosensor to evaluate the activity of putative TAAR1 ligands. This study represents the first comprehensive investigation of the membrane-expressed human TAAR1 pharmacology. Our strategy to express TAARs and to identify their ligands using a cAMP BRET assay could provide a foundation for characterizing the functional role of trace amines in vivo and suggests a strategy to apply to groups of poorly expressing GPCRs that have remained difficult to investigate in model systems.

Authors
Barak, LS; Salahpour, A; Zhang, X; Masri, B; Sotnikova, TD; Ramsey, AJ; Violin, JD; Lefkowitz, RJ; Caron, MG; Gainetdinov, RR
MLA Citation
Barak, LS, Salahpour, A, Zhang, X, Masri, B, Sotnikova, TD, Ramsey, AJ, Violin, JD, Lefkowitz, RJ, Caron, MG, and Gainetdinov, RR. "Pharmacological characterization of membrane-expressed human trace amine-associated receptor 1 (TAAR1) by a bioluminescence resonance energy transfer cAMP biosensor." Mol Pharmacol 74.3 (September 2008): 585-594.
PMID
18524885
Source
pubmed
Published In
Molecular pharmacology
Volume
74
Issue
3
Publish Date
2008
Start Page
585
End Page
594
DOI
10.1124/mol.108.048884

S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking.

Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that beta-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of beta-arrestin 2 is promoted by endogenous S-nitrosogluthathione. S-nitrosylation after agonist stimulation of the beta-adrenergic receptor, a prototypical GPCR, dissociates eNOS from beta-arrestin 2 and promotes binding of beta-arrestin 2 to clathrin heavy chain/beta-adaptin, thereby accelerating receptor internalization. The agonist- and NO-dependent shift in the affiliations of beta-arrestin 2 is followed by denitrosylation. Thus, beta-arrestin subserves the functional coupling of eNOS and GPCRs, and dynamic S-nitrosylation/denitrosylation of beta-arrestin 2 regulates stimulus-induced GPCR trafficking.

Authors
Ozawa, K; Whalen, EJ; Nelson, CD; Mu, Y; Hess, DT; Lefkowitz, RJ; Stamler, JS
MLA Citation
Ozawa, K, Whalen, EJ, Nelson, CD, Mu, Y, Hess, DT, Lefkowitz, RJ, and Stamler, JS. "S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking." Mol Cell 31.3 (August 8, 2008): 395-405.
PMID
18691971
Source
pubmed
Published In
Molecular Cell
Volume
31
Issue
3
Publish Date
2008
Start Page
395
End Page
405
DOI
10.1016/j.molcel.2008.05.024

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor.

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.

Authors
Nelson, CD; Kovacs, JJ; Nobles, KN; Whalen, EJ; Lefkowitz, RJ
MLA Citation
Nelson, CD, Kovacs, JJ, Nobles, KN, Whalen, EJ, and Lefkowitz, RJ. "Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor." J Biol Chem 283.30 (July 25, 2008): 21093-21101.
PMID
18534983
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
30
Publish Date
2008
Start Page
21093
End Page
21101
DOI
10.1074/jbc.M800431200

Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

Beta-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct beta-arrestin-mediated signaling. The recent discovery of beta-arrestin-biased ligands and receptor mutants has allowed characterization of these independent "G protein-mediated" and "beta-arrestin-mediated" signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of beta-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of beta-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that beta-arrestin can adopt multiple "active" conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in beta-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in beta-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling--i.e., functionally specific receptor conformations can be translated to downstream effectors such as beta-arrestins, thereby governing their functional specificity.

Authors
Shukla, AK; Violin, JD; Whalen, EJ; Gesty-Palmer, D; Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shukla, AK, Violin, JD, Whalen, EJ, Gesty-Palmer, D, Shenoy, SK, and Lefkowitz, RJ. "Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors." Proc Natl Acad Sci U S A 105.29 (July 22, 2008): 9988-9993.
PMID
18621717
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
105
Issue
29
Publish Date
2008
Start Page
9988
End Page
9993
DOI
10.1073/pnas.0804246105

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Authors
Kim, J; Zhang, L; Peppel, K; Wu, J-H; Zidar, DA; Brian, L; DeWire, SM; Exum, ST; Lefkowitz, RJ; Freedman, NJ
MLA Citation
Kim, J, Zhang, L, Peppel, K, Wu, J-H, Zidar, DA, Brian, L, DeWire, SM, Exum, ST, Lefkowitz, RJ, and Freedman, NJ. "Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration." Circ Res 103.1 (July 3, 2008): 70-79.
Website
http://hdl.handle.net/10161/5911
PMID
18519945
Source
pubmed
Published In
Circulation Research
Volume
103
Issue
1
Publish Date
2008
Start Page
70
End Page
79
DOI
10.1161/CIRCRESAHA.108.172338

Beta-arrestin-mediated localization of smoothened to the primary cilium.

beta-Arrestins have important roles in the regulation of seven-transmembrane receptors (7TMRs). Smoothened (Smo) is a 7TMR that mediates effects of Hedgehog on developmental processes and whose dysregulation may cause tumorigenesis. beta-Arrestins are required for endocytosis of Smo and signaling to Gli transcription factors. In mammalian cells, Smo-dependent signaling requires translocation to primary cilia. We demonstrated that beta-arrestins mediate the activity-dependent interaction of Smo and the kinesin motor protein Kif3A. This multimeric complex localized to primary cilia and was disrupted in cells transfected with beta-arrestin small interfering RNA. beta-Arrestin 1 or beta-arrestin 2 depletion prevented the localization of Smo to primary cilia and the Smo-dependent activation of Gli. These results suggest roles for beta-arrestins in mediating the intracellular transport of a 7TMR to its obligate subcellular location for signaling.

Authors
Kovacs, JJ; Whalen, EJ; Liu, R; Xiao, K; Kim, J; Chen, M; Wang, J; Chen, W; Lefkowitz, RJ
MLA Citation
Kovacs, JJ, Whalen, EJ, Liu, R, Xiao, K, Kim, J, Chen, M, Wang, J, Chen, W, and Lefkowitz, RJ. "Beta-arrestin-mediated localization of smoothened to the primary cilium." Science 320.5884 (June 27, 2008): 1777-1781.
PMID
18497258
Source
pubmed
Published In
Science
Volume
320
Issue
5884
Publish Date
2008
Start Page
1777
End Page
1781
DOI
10.1126/science.1157983

Crystallizing thinking about the beta2-adrenergic receptor.

Two recently determined crystal structures of the human beta2-adrenergic receptor (beta2AR) provide a long-awaited advance in the field of G protein-coupled receptor research. The beta2AR is only the second member of this, the largest family of receptors encoded in the human genome, whose structure has been solved. It follows structures of rhodopsin that were determined previously. Here we set these developments in historical context, discuss the daunting challenges that have been overcome, and appraise what has and has not been learned.

Authors
Shukla, AK; Sun, J-P; Lefkowitz, RJ
MLA Citation
Shukla, AK, Sun, J-P, and Lefkowitz, RJ. "Crystallizing thinking about the beta2-adrenergic receptor." Mol Pharmacol 73.5 (May 2008): 1333-1338.
PMID
18239031
Source
pubmed
Published In
Molecular pharmacology
Volume
73
Issue
5
Publish Date
2008
Start Page
1333
End Page
1338
DOI
10.1124/mol.108.045849

Beta-arrestin-mediated signaling regulates protein synthesis.

Seven transmembrane receptors (7TMRs) exert strong regulatory influences on virtually all physiological processes. Although it is historically assumed that heterotrimeric G proteins mediate these actions, there is a newer appreciation that beta-arrestins, originally thought only to desensitize G protein signaling, also serve as independent receptor signal transducers. Recently, we found that activation of ERK1/2 by the angiotensin receptor occurs via both of these distinct pathways. In this work, we explore the physiological consequences of beta-arrestin ERK1/2 signaling and delineate a pathway that regulates mRNA translation and protein synthesis via Mnk1, a protein that both physically interacts with and is activated by beta-arrestins. We show that beta-arrestin-dependent activation of ERK1/2, Mnk1, and eIF4E are responsible for increasing translation rates in both human embryonic kidney 293 and rat vascular smooth muscle cells. This novel demonstration that beta-arrestins regulate protein synthesis reveals that the spectrum of beta-arrestin-mediated signaling events is broader than previously imagined.

Authors
DeWire, SM; Kim, J; Whalen, EJ; Ahn, S; Chen, M; Lefkowitz, RJ
MLA Citation
DeWire, SM, Kim, J, Whalen, EJ, Ahn, S, Chen, M, and Lefkowitz, RJ. "Beta-arrestin-mediated signaling regulates protein synthesis." J Biol Chem 283.16 (April 18, 2008): 10611-10620.
PMID
18276584
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
16
Publish Date
2008
Start Page
10611
End Page
10620
DOI
10.1074/jbc.M710515200

The annual ASCI meeting: does nostalgia have a future?

For many academic physician-scientists, the yearly Tri-Societies meeting of the ASCI, AAP, and AFCR during the 1960s, '70s, and '80s was an annual rite of spring and the focal point of the academic year. In this brief essay, I set down some miscellaneous recollections of these meetings and some thoughts about why they were of such central importance in the careers of those of my generation.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "The annual ASCI meeting: does nostalgia have a future?." J Clin Invest 118.4 (April 2008): 1231-1233.
Website
http://hdl.handle.net/10161/7789
PMID
18382732
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
118
Issue
4
Publish Date
2008
Start Page
1231
End Page
1233
DOI
10.1172/JCI35024

beta-arrestin-biased agonism at the beta2-adrenergic receptor.

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.

Authors
Drake, MT; Violin, JD; Whalen, EJ; Wisler, JW; Shenoy, SK; Lefkowitz, RJ
MLA Citation
Drake, MT, Violin, JD, Whalen, EJ, Wisler, JW, Shenoy, SK, and Lefkowitz, RJ. "beta-arrestin-biased agonism at the beta2-adrenergic receptor." J Biol Chem 283.9 (February 29, 2008): 5669-5676.
PMID
18086673
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
9
Publish Date
2008
Start Page
5669
End Page
5676
DOI
10.1074/jbc.M708118200

beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics.

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Authors
Violin, JD; DiPilato, LM; Yildirim, N; Elston, TC; Zhang, J; Lefkowitz, RJ
MLA Citation
Violin, JD, DiPilato, LM, Yildirim, N, Elston, TC, Zhang, J, and Lefkowitz, RJ. "beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics." J Biol Chem 283.5 (February 1, 2008): 2949-2961.
PMID
18045878
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
5
Publish Date
2008
Start Page
2949
End Page
2961
DOI
10.1074/jbc.M707009200

β2-Adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous β2-adrenergic receptor (β2AR) in living cells. β2AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of ∼10 μM and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and β-arrestin to the regulation of β2AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to β-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of β2AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/β-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Violin, JD; DiPilato, LM; Yildirim, N; Elston, TC; Zhang, J; Lefkowitz, RJ
MLA Citation
Violin, JD, DiPilato, LM, Yildirim, N, Elston, TC, Zhang, J, and Lefkowitz, RJ. 2-Adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics." Journal of Biological Chemistry 283.5 (February 1, 2008): 2949-2961.
Source
scopus
Published In
The Journal of biological chemistry
Volume
283
Issue
5
Publish Date
2008
Start Page
2949
End Page
2961
DOI
10.1074/jbc.M707009200

A crystal clear view of the beta2-adrenergic receptor.

Authors
Lefkowitz, RJ; Sun, J-P; Shukla, AK
MLA Citation
Lefkowitz, RJ, Sun, J-P, and Shukla, AK. "A crystal clear view of the beta2-adrenergic receptor." Nat Biotechnol 26.2 (February 2008): 189-191.
PMID
18259173
Source
pubmed
Published In
Nature Biotechnology
Volume
26
Issue
2
Publish Date
2008
Start Page
189
End Page
191
DOI
10.1038/nbt0208-189

A beta-arrestin 2 signaling complex mediates lithium action on behavior.

Besides their role in desensitization, beta-arrestin 1 and 2 promote the formation of signaling complexes allowing G protein-coupled receptors (GPCR) to signal independently from G proteins. Here we show that lithium, a pharmacological agent used for the management of psychiatric disorders such as bipolar disorder, schizophrenia, and depression, regulates Akt/glycogen synthase kinase 3 (GSK3) signaling and related behaviors in mice by disrupting a signaling complex composed of Akt, beta-arrestin 2, and protein phosphatase 2A. When administered to beta-arrestin 2 knockout mice, lithium fails to affect Akt/GSK3 signaling and induce behavioral changes associated with GSK3 inhibition as it does in normal animals. These results point toward a pharmacological approach to modulating GPCR function that affects the formation of beta-arrestin-mediated signaling complexes.

Authors
Beaulieu, J-M; Marion, S; Rodriguiz, RM; Medvedev, IO; Sotnikova, TD; Ghisi, V; Wetsel, WC; Lefkowitz, RJ; Gainetdinov, RR; Caron, MG
MLA Citation
Beaulieu, J-M, Marion, S, Rodriguiz, RM, Medvedev, IO, Sotnikova, TD, Ghisi, V, Wetsel, WC, Lefkowitz, RJ, Gainetdinov, RR, and Caron, MG. "A beta-arrestin 2 signaling complex mediates lithium action on behavior." Cell 132.1 (January 11, 2008): 125-136.
PMID
18191226
Source
pubmed
Published In
Cell
Volume
132
Issue
1
Publish Date
2008
Start Page
125
End Page
136
DOI
10.1016/j.cell.2007.11.041

A beta-arrestin 2 signaling complex mediates lithium action on Behavior

Authors
Beaulieu, J-M; Marion, S; Rodriguiz, RM; Medvedev, IO; Sotnikova, TD; Ghisi, V; Wetsel, WC; Lefkowitz, RJ; Gainetdinov, RR; Caron, MG
MLA Citation
Beaulieu, J-M, Marion, S, Rodriguiz, RM, Medvedev, IO, Sotnikova, TD, Ghisi, V, Wetsel, WC, Lefkowitz, RJ, Gainetdinov, RR, and Caron, MG. "A beta-arrestin 2 signaling complex mediates lithium action on Behavior." CELL 132.1 (January 11, 2008): 125-136.
Source
wos-lite
Published In
Cell
Volume
132
Issue
1
Publish Date
2008
Start Page
125
End Page
136
DOI
10.1016/j.cell.2007.11.041

A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling.

For many years, beta-adrenergic receptor antagonists (beta-blockers or betaAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, beta-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different beta-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective betaAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant betaAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in beta2 adrenergic receptor (beta2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating G(s)-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of beta-arrestin to the beta2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant beta2AR(T68F,Y132G,Y219A) (beta2AR(TYY)) and abolished by beta-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from G(s), is nonetheless able to stimulate beta-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic beta2AR ligands.

Authors
Wisler, JW; DeWire, SM; Whalen, EJ; Violin, JD; Drake, MT; Ahn, S; Shenoy, SK; Lefkowitz, RJ
MLA Citation
Wisler, JW, DeWire, SM, Whalen, EJ, Violin, JD, Drake, MT, Ahn, S, Shenoy, SK, and Lefkowitz, RJ. "A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling." Proc Natl Acad Sci U S A 104.42 (October 16, 2007): 16657-16662.
PMID
17925438
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
42
Publish Date
2007
Start Page
16657
End Page
16662
DOI
10.1073/pnas.0707936104

Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation.

Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more. In cellular coimmunoprecipitation assays, beta-arrestin2(0K) bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, beta-arrestin2(0K) was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated beta(2)AR complexes. Although the wild type beta-arrestin2 formed ERK signaling complexes with the beta(2)AR at the membrane, a stably ubiquitinated beta-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of beta-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of beta-arrestin for membrane association. Our findings suggest that although beta-arrestin ubiquitination is dispensable for beta-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.

Authors
Shenoy, SK; Barak, LS; Xiao, K; Ahn, S; Berthouze, M; Shukla, AK; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Shenoy, SK, Barak, LS, Xiao, K, Ahn, S, Berthouze, M, Shukla, AK, Luttrell, LM, and Lefkowitz, RJ. "Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation." J Biol Chem 282.40 (October 5, 2007): 29549-29562.
PMID
17666399
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
40
Publish Date
2007
Start Page
29549
End Page
29562
DOI
10.1074/jbc.M700852200

Targeting hedgehog signaling in medulloblastoma

Authors
Wang, J; Liu, R; Bond, M; Chen, M; Singh, S; Ali-Osman, F; Lefkowitz, RJ; Diehl, AM; Lyerly, HK; Barak, L; Chen, W
MLA Citation
Wang, J, Liu, R, Bond, M, Chen, M, Singh, S, Ali-Osman, F, Lefkowitz, RJ, Diehl, AM, Lyerly, HK, Barak, L, and Chen, W. "Targeting hedgehog signaling in medulloblastoma." October 2007.
Source
wos-lite
Published In
Neuro-Oncology
Volume
9
Issue
4
Publish Date
2007
Start Page
560
End Page
561

Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection.

Deleterious effects on the heart from chronic stimulation of beta-adrenergic receptors (betaARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby beta-arrestins mediate beta1AR signaling to the EGFR. This beta-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via beta-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Authors
Noma, T; Lemaire, A; Naga Prasad, SV; Barki-Harrington, L; Tilley, DG; Chen, J; Le Corvoisier, P; Violin, JD; Wei, H; Lefkowitz, RJ; Rockman, HA
MLA Citation
Noma, T, Lemaire, A, Naga Prasad, SV, Barki-Harrington, L, Tilley, DG, Chen, J, Le Corvoisier, P, Violin, JD, Wei, H, Lefkowitz, RJ, and Rockman, HA. "Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection." J Clin Invest 117.9 (September 2007): 2445-2458.
Website
http://hdl.handle.net/10161/5925
PMID
17786238
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
117
Issue
9
Publish Date
2007
Start Page
2445
End Page
2458
DOI
10.1172/JCI31901

Beta-arrestin-biased ligands at seven-transmembrane receptors.

Seven-transmembrane receptors (7TMRs), the most common molecular targets of modern drug therapy, are critically regulated by beta-arrestins, which both inhibit classic G-protein signaling and initiate distinct beta-arrestin signaling. The interplay of G-protein and beta-arrestin signals largely determines the cellular consequences of 7TMR-targeted drugs. Until recently, a drug's efficacy for beta-arrestin recruitment was believed to be proportional to its efficacy for G-protein activities. This paradigm restricts 7TMR drug effects to a linear spectrum of responses, ranging from inhibition of all responses to stimulation of all responses. However, it is now clear that 'biased ligands' can selectively activate G-protein or beta-arrestin functions and thus elicit novel biological effects from even well-studied 7TMRs. Here, we discuss the current state of beta-arrestin-biased ligand research and the prospects for beta-arrestin bias as a therapeutic target. Consideration of ligand bias might have profound influences on the way scientists approach 7TMR-targeted drug discovery.

Authors
Violin, JD; Lefkowitz, RJ
MLA Citation
Violin, JD, and Lefkowitz, RJ. "Beta-arrestin-biased ligands at seven-transmembrane receptors." Trends Pharmacol Sci 28.8 (August 2007): 416-422. (Review)
PMID
17644195
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
28
Issue
8
Publish Date
2007
Start Page
416
End Page
422
DOI
10.1016/j.tips.2007.06.006

The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2.

beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.

Authors
Nobles, KN; Guan, Z; Xiao, K; Oas, TG; Lefkowitz, RJ
MLA Citation
Nobles, KN, Guan, Z, Xiao, K, Oas, TG, and Lefkowitz, RJ. "The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and -2." J Biol Chem 282.29 (July 20, 2007): 21370-21381.
PMID
17513300
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
29
Publish Date
2007
Start Page
21370
End Page
21381
DOI
10.1074/jbc.M611483200

Functional specialization of beta-arrestin interactions revealed by proteomic analysis.

Beta-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of beta-arrestins pertinent to these functions has been documented. The discovery of several novel functions of beta-arrestins stimulated us to perform a global proteomics analysis of beta-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with beta-arrestin 1, 164 interacted with beta-arrestin 2, and 102 interacted with both beta-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the beta-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with beta-arrestins. The beta-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified beta-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind beta-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.

Authors
Xiao, K; McClatchy, DB; Shukla, AK; Zhao, Y; Chen, M; Shenoy, SK; Yates, JR; Lefkowitz, RJ
MLA Citation
Xiao, K, McClatchy, DB, Shukla, AK, Zhao, Y, Chen, M, Shenoy, SK, Yates, JR, and Lefkowitz, RJ. "Functional specialization of beta-arrestin interactions revealed by proteomic analysis." Proc Natl Acad Sci U S A 104.29 (July 17, 2007): 12011-12016.
PMID
17620599
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
29
Publish Date
2007
Start Page
12011
End Page
12016
DOI
10.1073/pnas.0704849104

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

Authors
Whalen, EJ; Foster, MW; Matsumoto, A; Ozawa, K; Violin, JD; Que, LG; Nelson, CD; Benhar, M; Keys, JR; Rockman, HA; Koch, WJ; Daaka, Y; Lefkowitz, RJ; Stamler, JS
MLA Citation
Whalen, EJ, Foster, MW, Matsumoto, A, Ozawa, K, Violin, JD, Que, LG, Nelson, CD, Benhar, M, Keys, JR, Rockman, HA, Koch, WJ, Daaka, Y, Lefkowitz, RJ, and Stamler, JS. "Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2." Cell 129.3 (May 4, 2007): 511-522.
PMID
17482545
Source
pubmed
Published In
Cell
Volume
129
Issue
3
Publish Date
2007
Start Page
511
End Page
522
DOI
10.1016/j.cell.2007.02.046

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2

Authors
Whalen, EJ; Foster, MW; Matsumoto, A; Ozawa, K; Violin, JD; Que, LG; Nelson, CD; Benhar, M; Keys, JR; Rockman, HA; Koch, WJ; Daaka, Y; Lefkowitz, RJ; Stamler, JS
MLA Citation
Whalen, EJ, Foster, MW, Matsumoto, A, Ozawa, K, Violin, JD, Que, LG, Nelson, CD, Benhar, M, Keys, JR, Rockman, HA, Koch, WJ, Daaka, Y, Lefkowitz, RJ, and Stamler, JS. "Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2." CELL 129.3 (May 4, 2007): 511-522.
Source
wos-lite
Published In
Cell
Volume
129
Issue
3
Publish Date
2007
Start Page
511
End Page
522
DOI
10.1016/j.cell.2007.02.046

Seven transmembrane receptors: something old, something new.

Receptors for hormones, neurotransmitters, drugs, sensory stimuli and many other agents represent the gateway to cellular metabolism and activity. They regulate virtually all physiological processes in mammals. Yet as recently as 40 years ago their very existence was still in question. One class of receptors, those coupled to G proteins (also known as GPCRs or seven transmembrane receptors) comprise by far the largest group (approx. 1000), and are the most important target of clinically used drugs. Here I provide a very personal retrospective of research over the past 35 years which ultimately led to the identification, purification, reconstitution and cloning of the adrenergic receptors; the discovery of their homology with the seven transmembrane spanning visual light receptor rhodopsin and the realization that there was a large gene family of G protein coupled receptors; the elucidation of the molecular mechanisms of receptor desensitization and signalling through G protein-coupled receptor kinases and beta-arrestins; and the appreciation that the structure, signalling, and regulatory mechanisms of the receptors are all highly conserved across the large receptor superfamily.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Seven transmembrane receptors: something old, something new." Acta Physiol (Oxf) 190.1 (May 2007): 9-19. (Review)
PMID
17428228
Source
pubmed
Published In
Acta Physiologica
Volume
190
Issue
1
Publish Date
2007
Start Page
9
End Page
19
DOI
10.1111/j.1365-201X.2007.01693.x

Seven transmembrane receptors: something old, something new

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Seven transmembrane receptors: something old, something new." May 2007.
Source
wos-lite
Published In
Acta Physiologica
Volume
190
Issue
1
Publish Date
2007
Start Page
9
End Page
19
DOI
10.1111/j.1748-1716.2007.01693.x

Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression.

Beta-arrestin1, which regulates many aspects of seven transmembrane receptor (7TMR) biology, has also been shown to serve as an adaptor, which brings Mdm2, an E3 ubiquitin ligase to the insulin-like growth factor-1 receptor (IGF-1R), leading to its proteasome-dependent destruction. Here we demonstrate that IGF-1R stimulation also leads to ubiquitination of beta-arrestin1, which regulates vesicular trafficking and activation of ERK1/2. This beta-arrestin1-dependent ERK activity can occur even when the classical tyrosine kinase signaling is impaired. siRNA-mediated suppression of beta-arrestin1 in human melanoma cells ablates IGF-1-stimulated ERK and prolongs the G1 phase of the cell cycle. These data suggest that beta-arrestin-dependent ERK signaling by the IGF-1R regulates cell cycle progression and may thus be an important regulator of the growth of normal and malignant cells.

Authors
Girnita, L; Shenoy, SK; Sehat, B; Vasilcanu, R; Vasilcanu, D; Girnita, A; Lefkowitz, RJ; Larsson, O
MLA Citation
Girnita, L, Shenoy, SK, Sehat, B, Vasilcanu, R, Vasilcanu, D, Girnita, A, Lefkowitz, RJ, and Larsson, O. "Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression." J Biol Chem 282.15 (April 13, 2007): 11329-11338.
PMID
17303558
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
15
Publish Date
2007
Start Page
11329
End Page
11338
DOI
10.1074/jbc.M611526200

Seven transmembrane receptors: a brief personal retrospective.

Receptors have fascinated biologists for more than a century and they have fascinated me for the entirety of my own research career. The seven transmembrane receptors, also known as G protein coupled receptors, represent the largest of the several families of plasma membrane receptors, comprising more than a thousand genes and regulating virtually all known physiological processes in mammals. Moreover, they represent one of the commonest targets of currently used drugs. I have spent the entirety of my research career working on these receptors. Here I set down some personal reflections on the evolution of the field during the past 35 years, hanging the thread of the story on some of the work from my own laboratory.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Seven transmembrane receptors: a brief personal retrospective." Biochim Biophys Acta 1768.4 (April 2007): 748-755.
PMID
17173855
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1768
Issue
4
Publish Date
2007
Start Page
748
End Page
755
DOI
10.1016/j.bbamem.2006.11.001

Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins.

Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide-binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that beta-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. beta-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, beta-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the beta-arrestin-DGK complex to activated 7TMRs. The dual function of beta-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3',5'-monophosphate phosphodiesterases to Gs-coupled beta2-adrenergic receptors. Thus, beta-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.

Authors
Nelson, CD; Perry, SJ; Regier, DS; Prescott, SM; Topham, MK; Lefkowitz, RJ
MLA Citation
Nelson, CD, Perry, SJ, Regier, DS, Prescott, SM, Topham, MK, and Lefkowitz, RJ. "Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins." Science 315.5812 (February 2, 2007): 663-666.
PMID
17272726
Source
pubmed
Published In
Science
Volume
315
Issue
5812
Publish Date
2007
Start Page
663
End Page
666
DOI
10.1126/science.1134562

Beta-arrestins and cell signaling.

Upon their discovery, beta-arrestins 1 and 2 were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors, ultimately resulting in receptor desensitization. Surprisingly, recent evidence shows that beta-arrestins can also function to activate signaling cascades independently of G protein activation. By serving as multiprotein scaffolds, the beta-arrestins bring elements of specific signaling pathways into close proximity. beta-Arrestin regulation has been demonstrated for an ever-increasing number of signaling molecules, including the mitogen-activated protein kinases ERK, JNK, and p38 as well as Akt, PI3 kinase, and RhoA. In addition, investigators are discovering new roles for beta-arrestins in nuclear functions. Here, we review the signaling capacities of these versatile adapter molecules and discuss the possible implications for cellular processes such as chemotaxis and apoptosis.

Authors
DeWire, SM; Ahn, S; Lefkowitz, RJ; Shenoy, SK
MLA Citation
DeWire, SM, Ahn, S, Lefkowitz, RJ, and Shenoy, SK. "Beta-arrestins and cell signaling." Annu Rev Physiol 69 (2007): 483-510. (Review)
PMID
17305471
Source
pubmed
Published In
Annual Review of Physiology
Volume
69
Publish Date
2007
Start Page
483
End Page
510
DOI
10.1146/annurev.ph.69.013107.100021

β-Arrestins and cell signaling

Upon their discovery, β-arrestins 1 and 2 were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors, ultimately resulting in receptor desensitization. Surprisingly, recent evidence shows that β-arrestins can also function to activate signaling cascades independently of G protein activation. By serving as multiprotein scaffolds, the β-arrestins bring elements of specific signaling pathways into close proximity. β-Arrestin regulation has been demonstrated for an ever-increasing number of signaling molecules, including the mitogen-activated protein kinases ERK, JNK, and p38 as well as Akt, PI3 kinase, and RhoA. In addition, investigators are discovering new roles for β-arrestins in nuclear functions. Here, we review the signaling capacities of these versatile adapter molecules and discuss the possible implications for cellular processes such as chemotaxis and apoptosis. Copyright © 2007 by Annual Reviews. All rights reserved.

Authors
DeWire, SM; Ahn, S; Lefkowitz, RJ; Shenoy, SK
MLA Citation
DeWire, SM, Ahn, S, Lefkowitz, RJ, and Shenoy, SK. "β-Arrestins and cell signaling." Annual Review of Physiology 69 (2007): 483-510.
Source
scival
Published In
Annual Review of Physiology
Volume
69
Publish Date
2007
Start Page
483
End Page
510
DOI
10.1146/annurev.physiol.69.022405.154749

Introduction to special section on β-arrestins

Authors
Lefkowitz, R
MLA Citation
Lefkowitz, R. "Introduction to special section on β-arrestins." Annual Review of Physiology 69 (2007): xi-xii.
Source
scival
Published In
Annual Review of Physiology
Volume
69
Publish Date
2007
Start Page
xi
End Page
xii

New roles for beta-arrestins in cell signaling: not just for seven-transmembrane receptors.

beta-arrestins, originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of the G protein-coupled beta2-adrenergic receptor (beta2-AR), have recently emerged as multifunctional adaptor/scaffold proteins that dynamically assemble a wide range of multiprotein complexes in response to stimulation of most seven-transmembrane receptors (7TMRs). These complexes mediate receptor signaling, trafficking, and degradation. Moreover, beta-arrestins are increasingly found to perform analogous functions for receptors from structurally diverse classes, including atypical 7TMRs such as frizzled and smoothened, the nicotinic cholinergic receptors, receptor tyrosine kinases, and cytokine receptors, thereby regulating a growing list of cellular processes such as chemotaxis, apoptosis, and metastasis.

Authors
Lefkowitz, RJ; Rajagopal, K; Whalen, EJ
MLA Citation
Lefkowitz, RJ, Rajagopal, K, and Whalen, EJ. "New roles for beta-arrestins in cell signaling: not just for seven-transmembrane receptors." Mol Cell 24.5 (December 8, 2006): 643-652. (Review)
PMID
17157248
Source
pubmed
Published In
Molecular Cell
Volume
24
Issue
5
Publish Date
2006
Start Page
643
End Page
652
DOI
10.1016/j.molcel.2006.11.007

G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

Authors
Violin, JD; Dewire, SM; Barnes, WG; Lefkowitz, RJ
MLA Citation
Violin, JD, Dewire, SM, Barnes, WG, and Lefkowitz, RJ. "G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics." J Biol Chem 281.47 (November 24, 2006): 36411-36419.
PMID
17008309
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
47
Publish Date
2006
Start Page
36411
End Page
36419
DOI
10.1074/jbc.M607956200

Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes.

The G protein-coupled receptor kinases (GRKs) and beta-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/beta-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/beta-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT(1A)R) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the "biased" AT(1A)R agonist [Sar(1), Ile(4), Ile(8)]-angiotensin II (SII), which is unable to stimulate G(alpha)q-mediated signaling, but which has previously been shown to promote beta-arrestin interaction with the AT(1A)R. Cardiomyocytes from WT, but not AT(1A)R-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from beta-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2(+/-)) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent G(alpha)q/PKC- and GRK6/beta-arrestin2-dependent mechanisms by which stimulation of the AT(1A)R can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.

Authors
Rajagopal, K; Whalen, EJ; Violin, JD; Stiber, JA; Rosenberg, PB; Premont, RT; Coffman, TM; Rockman, HA; Lefkowitz, RJ
MLA Citation
Rajagopal, K, Whalen, EJ, Violin, JD, Stiber, JA, Rosenberg, PB, Premont, RT, Coffman, TM, Rockman, HA, and Lefkowitz, RJ. "Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes." Proc Natl Acad Sci U S A 103.44 (October 31, 2006): 16284-16289.
PMID
17060617
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
44
Publish Date
2006
Start Page
16284
End Page
16289
DOI
10.1073/pnas.0607583103

beta-arrestin mediated beta 1-adrenergic receptor transactivartion of the epidermal growth factor receptor requires GRK 5 and 6

Authors
Chen, J; Naga Prasad, SV; Tilley, DG; Lefkowitz, RJ; Rockman, HA
MLA Citation
Chen, J, Naga Prasad, SV, Tilley, DG, Lefkowitz, RJ, and Rockman, HA. "beta-arrestin mediated beta 1-adrenergic receptor transactivartion of the epidermal growth factor receptor requires GRK 5 and 6." October 31, 2006.
Source
wos-lite
Published In
Circulation
Volume
114
Issue
18
Publish Date
2006
Start Page
159
End Page
159

beta-arrestins regulate proliferation and migration of smooth muscle cells in vitro & in vivo: Isoform-specific roles

Authors
Kim, J; Zhang, L; Peppel, K; Lefkowitz, RJ; Freedman, NJ
MLA Citation
Kim, J, Zhang, L, Peppel, K, Lefkowitz, RJ, and Freedman, NJ. "beta-arrestins regulate proliferation and migration of smooth muscle cells in vitro & in vivo: Isoform-specific roles." October 31, 2006.
Source
wos-lite
Published In
Circulation
Volume
114
Issue
18
Publish Date
2006
Start Page
296
End Page
296

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

Authors
Meloni, AR; Fralish, GB; Kelly, P; Salahpour, A; Chen, JK; Wechsler-Reya, RJ; Lefkowitz, RJ; Caron, MG
MLA Citation
Meloni, AR, Fralish, GB, Kelly, P, Salahpour, A, Chen, JK, Wechsler-Reya, RJ, Lefkowitz, RJ, and Caron, MG. "Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2." Mol Cell Biol 26.20 (October 2006): 7550-7560.
Website
http://hdl.handle.net/10161/7792
PMID
16908539
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
26
Issue
20
Publish Date
2006
Start Page
7550
End Page
7560
DOI
10.1128/MCB.00546-06

Trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

Authors
Drake, MT; Shenoy, SK; Lefkowitz, RJ
MLA Citation
Drake, MT, Shenoy, SK, and Lefkowitz, RJ. "Trafficking of G protein-coupled receptors." Circ Res 99.6 (September 15, 2006): 570-582.
Website
http://hdl.handle.net/10161/5934
PMID
16973913
Source
pubmed
Published In
Circulation Research
Volume
99
Issue
6
Publish Date
2006
Start Page
570
End Page
582
DOI
10.1161/01.RES.0000242563.47507.ce

Beta-arrestin-dependent signaling by seven-transmembrane receptors

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Beta-arrestin-dependent signaling by seven-transmembrane receptors." August 30, 2006.
Source
wos-lite
Published In
Regulatory Peptides
Volume
135
Issue
3
Publish Date
2006
Start Page
102
End Page
102

G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer.

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.

Authors
Violin, JD; Ren, X-R; Lefkowitz, RJ
MLA Citation
Violin, JD, Ren, X-R, and Lefkowitz, RJ. "G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer." J Biol Chem 281.29 (July 21, 2006): 20577-20588.
PMID
16687412
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
29
Publish Date
2006
Start Page
20577
End Page
20588
DOI
10.1074/jbc.M513605200

GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling.

Stimulation of cell-surface seven-transmembrane receptors (7TMRs) elicits biological responses to a wide range of extracellular signals, including many hormones. Classically, heterotrimeric GTP-binding proteins (G proteins) are recruited to the activated conformation of 7TMRs. Only two other families of protein have this remarkable characteristic: G-protein-coupled receptor kinases and beta-arrestins. These two protein families have long been known to have a central and coordinated role in the "desensitization" of G protein activation by 7TMRs. In addition, G-protein-coupled receptor kinases and beta-arrestins are involved in an increasing number of interactions with non-receptor proteins, broadening the variety of their cellular functions. These newly appreciated attributes of these two families of protein highlight their unique ability to coordinate the various aspects of 7TMR functions.

Authors
Reiter, E; Lefkowitz, RJ
MLA Citation
Reiter, E, and Lefkowitz, RJ. "GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling." Trends Endocrinol Metab 17.4 (May 2006): 159-165. (Review)
PMID
16595179
Source
pubmed
Published In
Trends in Endocrinology and Metabolism
Volume
17
Issue
4
Publish Date
2006
Start Page
159
End Page
165
DOI
10.1016/j.tem.2006.03.008

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Authors
Gesty-Palmer, D; Chen, M; Reiter, E; Ahn, S; Nelson, CD; Wang, S; Eckhardt, AE; Cowan, CL; Spurney, RF; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Gesty-Palmer, D, Chen, M, Reiter, E, Ahn, S, Nelson, CD, Wang, S, Eckhardt, AE, Cowan, CL, Spurney, RF, Luttrell, LM, and Lefkowitz, RJ. "Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation." J Biol Chem 281.16 (April 21, 2006): 10856-10864.
PMID
16492667
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
16
Publish Date
2006
Start Page
10856
End Page
10864
DOI
10.1074/jbc.M513380200

Conformational changes in beta-arrestin1: The importance of beta-arrestin1's N-domain

Authors
Nobles, KN; Guan, ZQ; Xiao, KH; Oas, TG; Lefkowitz, RJ
MLA Citation
Nobles, KN, Guan, ZQ, Xiao, KH, Oas, TG, and Lefkowitz, RJ. "Conformational changes in beta-arrestin1: The importance of beta-arrestin1's N-domain." March 6, 2006.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
20
Issue
4
Publish Date
2006
Start Page
A114
End Page
A114

beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor.

Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.

Authors
Shenoy, SK; Drake, MT; Nelson, CD; Houtz, DA; Xiao, K; Madabushi, S; Reiter, E; Premont, RT; Lichtarge, O; Lefkowitz, RJ
MLA Citation
Shenoy, SK, Drake, MT, Nelson, CD, Houtz, DA, Xiao, K, Madabushi, S, Reiter, E, Premont, RT, Lichtarge, O, and Lefkowitz, RJ. "beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor." J Biol Chem 281.2 (January 13, 2006): 1261-1273.
PMID
16280323
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
2
Publish Date
2006
Start Page
1261
End Page
1273
DOI
10.1074/jbc.M506576200

Receptor regulation: β-arrestin moves up a notch

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Receptor regulation: β-arrestin moves up a notch." Nature Cell Biology 7.12 (December 2005): 1159-1161.
Source
crossref
Published In
Nature Cell Biology
Volume
7
Issue
12
Publish Date
2005
Start Page
1159
End Page
1161
DOI
10.1038/ncb1205-1159

Receptor regulation: beta-arrestin moves up a notch.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Receptor regulation: beta-arrestin moves up a notch." Nat Cell Biol 7.12 (December 2005): 1159-1161.
PMID
16319967
Source
pubmed
Published In
Nature Cell Biology
Volume
7
Issue
12
Publish Date
2005
Start Page
1159
End Page
1161
DOI
10.1038/ncb1205-1059

Angiotensin II-stimulated signaling through G proteins and beta-arrestin.

Beta-arrestin, originally identified as a protein that inhibits heterotrimeric guanine nucleotide-binding protein (G protein) coupling to cognate seven-transmembrane receptors [(7TMRs), also known as G protein-coupled receptors (GPCRs)], is currently being appreciated as a positive signaling mediator for various cell surface receptors. Activation of mitogen-activated protein kinases (MAPKs), especially extracellular signal regulated kinases 1 and 2 (ERK1/2), is a hallmark of intracellular signaling resulting from stimulation of various growth factor receptors, as well as 7TMRs. The resulting ERK activity can occur through multiple parallel or converging mechanisms. Using human embryonic kidney 293 (HEK-293) cells as a model system and utilizing RNA interference technology, two distinct pathways of angiotensin II-mediated ERK activation have been uncovered: (i) a G protein-dependent pathway that produces a transient activation of nuclear ERK and (ii) a beta-arrestin-dependent pathway that leads to sustained activation of ERK that is localized to the cytosol and endosomes. The spatial and temporal segregation of ERK activated by G protein and beta-arrestin pathways suggests that the physiological consequences may be different, and thus ligands that selectively stimulate or inhibit one of these pathways may be therapeutically valuable.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Angiotensin II-stimulated signaling through G proteins and beta-arrestin. (Published online)" Sci STKE 2005.311 (November 22, 2005): cm14-.
PMID
16304060
Source
pubmed
Published In
Sciences STKE [electronic resource] : signal transduction knowledge environment
Volume
2005
Issue
311
Publish Date
2005
Start Page
cm14
DOI
10.1126/stke.3112005cm14

Seven-transmembrane receptor signaling through beta-arrestin.

Cell surface receptors are important communicators of external stimuli to the cell interior where they lead to initiation of various signaling pathways and cellular responses. The largest receptor family is the seven-transmembrane receptor (7TMR) family, with approximately 1000 coding genes in the human genome. When 7TMRs are stimulated with agonists, they activate heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to the production of signaling second messengers, such as adenosine 3',5'-monophosphate, inositol phosphates, and others. Activated receptors are rapidly phosphorylated on serine and threonine residues by specialized enzymes called G protein-coupled receptor kinases. Phosphorylated receptors bind the multifunctional adaptor proteins beta-arrestin1 and beta-arrestin2 with high affinity. Beta-arrestin binding blocks further G protein coupling, leading to "desensitization" of G protein-dependent signaling pathways. For several years, this was considered the sole function of beta-arrestins. However, novel functions of beta-arrestins have been discovered. Beta-arrestins are now designated as important adaptors that link receptors to the clathrin-dependent pathway of internalization. Beta-arrestins bind and direct the activity of several nonreceptor tyrosine kinases in response to 7TMR stimulation. Beta-arrestins also bind and scaffold members of such signaling cascades as the mitogen-activated protein kinases (MAPKs). Beta-arrestins are crucial components in 7TMR signaling leading to cellular responses that include cell survival and chemotaxis. Beta-arrestins act as endocytic adaptors and signal mediators not only for the 7TMRs, but also for several receptor tyrosine kinases.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Seven-transmembrane receptor signaling through beta-arrestin. (Published online)" Sci STKE 2005.308 (November 1, 2005): cm10-. (Review)
PMID
16267056
Source
pubmed
Published In
Sciences STKE [electronic resource] : signal transduction knowledge environment
Volume
2005
Issue
308
Publish Date
2005
Start Page
cm10
DOI
10.1126/stke.2005/308/cm10

When 7 transmembrane receptors are not G protein-coupled receptors.

Classically, 7 transmembrane receptors transduce extracellular signals by coupling to heterotrimeric G proteins, although recent in vitro studies have clearly demonstrated that they can also signal via G protein-independent mechanisms. However, the physiologic consequences of this unconventional signaling, particularly in vivo, have not been explored. In this issue of the JCI, Zhai et al. demonstrate in vivo effects of G protein-independent signaling by the angiotensin II type 1 receptor (AT1R) (see the related article beginning on page 3045). In studies of the mouse heart, they compare the physiologic and biochemical consequences of transgenic cardiac-specific overexpression of a mutant AT1R incapable of G protein coupling with those of a wild-type receptor. Their results not only provide the first glimpse of the physiologic effects of this newly appreciated mode of signaling but also provide important and previously unappreciated clues as to the underlying molecular mechanisms.

Authors
Rajagopal, K; Lefkowitz, RJ; Rockman, HA
MLA Citation
Rajagopal, K, Lefkowitz, RJ, and Rockman, HA. "When 7 transmembrane receptors are not G protein-coupled receptors." J Clin Invest 115.11 (November 2005): 2971-2974. (Review)
Website
http://hdl.handle.net/10161/5931
PMID
16276410
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
115
Issue
11
Publish Date
2005
Start Page
2971
End Page
2974
DOI
10.1172/JCI26950

beta-arrestin-dependent, G-protein-independent ERK1/2 activation by the beta(2)-adrenergic receptor

Authors
Shenoy, SK; Drake, MT; Nelson, CD; Houtz, DA; Xiao, KH; Madabushi, S; Reiter, E; Premont, RT; Lichtarge, O; Lefkowitz, RJ
MLA Citation
Shenoy, SK, Drake, MT, Nelson, CD, Houtz, DA, Xiao, KH, Madabushi, S, Reiter, E, Premont, RT, Lichtarge, O, and Lefkowitz, RJ. "beta-arrestin-dependent, G-protein-independent ERK1/2 activation by the beta(2)-adrenergic receptor." October 25, 2005.
Source
wos-lite
Published In
Circulation
Volume
112
Issue
17
Publish Date
2005
Start Page
U26
End Page
U26

Beta-arrestin 2-dependent positive inotropic and lusitropic responses to angiotensin II receptor stimulation in cardiomyocytes

Authors
Rajagopal, K; Whalen, EJ; Violin, JD; Rockman, HA; Lefkowitz, RJ
MLA Citation
Rajagopal, K, Whalen, EJ, Violin, JD, Rockman, HA, and Lefkowitz, RJ. "Beta-arrestin 2-dependent positive inotropic and lusitropic responses to angiotensin II receptor stimulation in cardiomyocytes." October 25, 2005.
Source
wos-lite
Published In
Circulation
Volume
112
Issue
17
Publish Date
2005
Start Page
U237
End Page
U237

beta-arrestin dependent, G protein independent ERIK1/2 activation by the beta 2 adrenergic receptor

Authors
Shenoy, SK; Drake, MT; Nelson, CD; Houtz, DA; Xiao, KH; Madabushi, S; Reiter, E; Premont, RT; Lichtarge, O; Lefkowitz, RJ
MLA Citation
Shenoy, SK, Drake, MT, Nelson, CD, Houtz, DA, Xiao, KH, Madabushi, S, Reiter, E, Premont, RT, Lichtarge, O, and Lefkowitz, RJ. "beta-arrestin dependent, G protein independent ERIK1/2 activation by the beta 2 adrenergic receptor." October 25, 2005.
Source
wos-lite
Published In
Circulation
Volume
112
Issue
17
Publish Date
2005
Start Page
U47
End Page
U47

Distinct conformations of the parathyroid hormone receptor mediate G protein and beta-arrestin dependent activation of ERK1/2.

Authors
Gesty-Palmer, D; Reiter, E; Spurney, RF; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Gesty-Palmer, D, Reiter, E, Spurney, RF, Luttrell, LM, and Lefkowitz, RJ. "Distinct conformations of the parathyroid hormone receptor mediate G protein and beta-arrestin dependent activation of ERK1/2." September 2005.
Source
wos-lite
Published In
Journal of Bone and Mineral Research
Volume
20
Issue
9
Publish Date
2005
Start Page
S182
End Page
S182

An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior.

Dopamine plays an important role in the etiology of schizophrenia, and D2 class dopamine receptors are the best-established target of antipsychotic drugs. Here we show that D2 class-receptor-mediated Akt regulation involves the formation of signaling complexes containing beta-arrestin 2, PP2A, and Akt. beta-arrestin 2 deficiency in mice results in reduction of dopamine-dependent behaviors, loss of Akt regulation by dopamine in the striatum, and disruption of the dopamine-dependent interaction of Akt with its negative regulator, protein phosphatase 2A. Importantly, canonical cAMP-mediated dopamine-receptor signaling is not inhibited in the absence of beta-arrestin 2. These results demonstrate that, apart from its classical function in receptor desensitization, beta-arrestin 2 also acts as a signaling intermediate through a kinase/phosphatase scaffold. Furthermore, this function of beta-arrestin 2 is important for the expression of dopamine-associated behaviors, thus implicating beta-arrestin 2 as a positive mediator of dopaminergic synaptic transmission and a potential pharmacological target for dopamine-related psychiatric disorders.

Authors
Beaulieu, J-M; Sotnikova, TD; Marion, S; Lefkowitz, RJ; Gainetdinov, RR; Caron, MG
MLA Citation
Beaulieu, J-M, Sotnikova, TD, Marion, S, Lefkowitz, RJ, Gainetdinov, RR, and Caron, MG. "An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior." Cell 122.2 (July 29, 2005): 261-273.
PMID
16051150
Source
pubmed
Published In
Cell
Volume
122
Issue
2
Publish Date
2005
Start Page
261
End Page
273
DOI
10.1016/j.cell.2005.05.012

{beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase.

The insulin-like growth factor-1 receptor (IGF-1R) plays important roles in physiological growth and aging as well as promoting several crucial functions in cancer cells. However, the molecular mechanisms involved in expression and down-regulation of IGF-1R are still poorly understood. Here we provide evidence that beta-arrestin, otherwise known to be involved in the regulation of G protein-coupled receptors, serves as an adaptor to bring the oncoprotein E3 ubiquitin ligase MDM2 to the IGF-1R. In this way, beta-arrestin acts as a crucial component in the ubiquitination and down-regulation of the receptor. Both MDM2 and beta-arrestin co-immunoprecipitated with the IGF-1R. The beta-arrestin isoform 1 appeared to be more strongly associated with the receptor than isoform 2, and in a molecular context it was 4-fold more efficient in inducing polyubiquitination of IGF-1R, a reaction that required the presence of beta-arrestin and MDM2. Ligand stimulation accelerated IGF-1R ubiquitination. In mouse P6 cells (overexpressing human IGF-1R) absence of beta-arrestin 1, but not of beta-arrestin 2, blocked ubiquitination of IGF-1R. Conversely, in the two studied human melanoma cell lines both beta-arrestin isoforms seemed to be involved in IGF-1R ubiquitination. However, because depletion of beta-arrestin 1 almost completely eliminated degradation, and IGF-1 induced down-regulation of the receptor in these cells, whereas beta-arrestin 2 only had a partial effect, beta-arrestin 1 seems to the more important isoform in affecting the expression of IGF-1R. To our knowledge this is the first study demonstrating a defined molecular role of beta-arrestin with direct relevance to cell growth and cancer.

Authors
Girnita, L; Shenoy, SK; Sehat, B; Vasilcanu, R; Girnita, A; Lefkowitz, RJ; Larsson, O
MLA Citation
Girnita, L, Shenoy, SK, Sehat, B, Vasilcanu, R, Girnita, A, Lefkowitz, RJ, and Larsson, O. "{beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase." J Biol Chem 280.26 (July 1, 2005): 24412-24419.
PMID
15878855
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
26
Publish Date
2005
Start Page
24412
End Page
24419
DOI
10.1074/jbc.M501129200

Transduction of receptor signals by beta-arrestins.

The transmission of extracellular signals to the interior of the cell is a function of plasma membrane receptors, of which the seven transmembrane receptor family is by far the largest and most versatile. Classically, these receptors stimulate heterotrimeric G proteins, which control rates of generation of diffusible second messengers and entry of ions at the plasma membrane. Recent evidence, however, indicates another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways. They direct the recruitment, activation, and scaffolding of cytoplasmic signaling complexes via two multifunctional adaptor and transducer molecules, beta-arrestins 1 and 2. This mechanism regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.

Authors
Lefkowitz, RJ; Shenoy, SK
MLA Citation
Lefkowitz, RJ, and Shenoy, SK. "Transduction of receptor signals by beta-arrestins." Science 308.5721 (April 22, 2005): 512-517. (Review)
PMID
15845844
Source
pubmed
Published In
Science
Volume
308
Issue
5721
Publish Date
2005
Start Page
512
End Page
517
DOI
10.1126/science.1109237

Receptor-specific ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes.

Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, beta-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. Recently, the co-trafficking of receptor-beta-arrestin complexes to endosomes was demonstrated to require stable beta-arrestin ubiquitination (Shenoy, S. K., and Lefkowitz, R. J. (2003) J. Biol. Chem. 278, 14498-14506). We now report that lysines at positions 11 and 12 in beta-arrestin2 are specific and required sites for its AngII-mediated sustained ubiquitination. Thus, upon AngII stimulation the mutant beta-arrestin2(K11,12R) is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to beta-arrestin2(K11,12R) restores AngII-mediated trafficking and signaling. Wild type beta-arrestin2 and beta-arrestin2(K11R,K12R)-Ub, but not beta-arrestin2(K11R,K12R), prevent nuclear translocation of pERK. These findings imply that sustained beta-arrestin ubiquitination not only directs co-trafficking of receptor-beta-arrestin complexes but also orchestrates the targeting of "7TMR signalosomes" to microcompartments within the cell. Surprisingly, binding of beta-arrestin2(K11R,K12R) to V2R and NK1R is indistinguishable from that of wild type beta-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of beta-arrestin2(K11,12R) are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant (beta-arrestin2(K18R,K107R,K108R,K207R,K296R)) is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. Our findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of beta-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of beta-arrestin2.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Receptor-specific ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes." J Biol Chem 280.15 (April 15, 2005): 15315-15324.
PMID
15699045
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
15
Publish Date
2005
Start Page
15315
End Page
15324
DOI
10.1074/jbc.M412418200

Beta-arrestin 2-dependent angiotensin II type 1A receptor-mediated pathway of chemotaxis.

Chemotaxis is a cellular response that directs cell migration toward a chemical gradient and is fundamental to a variety of cellular processes. The receptors for most known chemokines belong to the seven transmembrane-spanning superfamily and signal through members of the G(alphai) family. Beta-arrestins, in addition to regulating desensitization, have emerged as potential mediators of G-protein-independent signaling pathways and have been implicated in several chemotactic pathways. Here, we report a system wherein chemotaxis is stimulated in a beta-arrestin 2-dependent and apparently G-protein-independent manner. Human embryonic kidney 293 cells with stable expression of the angiotensin II (Ang II) receptor type 1A (AT(1A)R) undergo chemotaxis in response to Ang II. An Ang II peptide analog S(1)I(4)I(8) Ang II that is unable to activate G-protein-mediated responses induces chemotaxis in these cells that is unaffected by pertussis toxin-mediated suppression of G(alphai). Suppression of beta-arrestin 2 expression using small interfering RNA (siRNA) essentially eliminated AT(1A)R-mediated chemotaxis induced by either Ang II or the S(1)I(4)I(8) Ang II peptide but had no effect on epidermal growth factor (EGF)-induced chemotaxis. It also abolished chemotaxis induced by lysophosphatidic acid (LPA), which was completely sensitive to pertussis toxin. In contrast, reduction of G(alphaq/11) through siRNA and inhibition of protein kinase C, extracellular signal-regulated kinases 1 and 2, or phosphatidylinositol-3-kinase did not diminish AT(1A)R-mediated chemotaxis. Inhibiting p38 mitogen-activated protein kinase decreased AT(1A)R-mediated chemotaxis and eliminated EGF-mediated chemotaxis, suggesting that p38 plays a role in chemotaxis that is not specific to the AT(1A)R in this system. These data suggest that beta-arrestin 2 can mediate chemotaxis through mechanisms which may be G-protein-independent (Ang II receptors) or -dependent (LPA receptors).

Authors
Hunton, DL; Barnes, WG; Kim, J; Ren, X-R; Violin, JD; Reiter, E; Milligan, G; Patel, DD; Lefkowitz, RJ
MLA Citation
Hunton, DL, Barnes, WG, Kim, J, Ren, X-R, Violin, JD, Reiter, E, Milligan, G, Patel, DD, and Lefkowitz, RJ. "Beta-arrestin 2-dependent angiotensin II type 1A receptor-mediated pathway of chemotaxis." Mol Pharmacol 67.4 (April 2005): 1229-1236.
PMID
15635042
Source
pubmed
Published In
Molecular pharmacology
Volume
67
Issue
4
Publish Date
2005
Start Page
1229
End Page
1236
DOI
10.1124/mol.104.006270

beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation.

beta-Arrestins were initially shown, in conjunction with G protein-coupled receptor kinases, to be involved in the desensitization and internalization of activated seven-transmembrane receptors. Recently, beta-arrestin 2 has been shown to act as a signal mediator in mitogen-activated protein kinase cascades and to play a positive regulatory role in chemotaxis. We now show that beta-arrestin 1 is required to activate the small GTPase RhoA leading to the re-organization of stress fibers following the activation of the angiotensin II type 1A receptor. This angiotensin II type 1A receptor-directed RhoA activation and stress fiber formation also require the activation of the heterotrimeric G protein G(alphaq/11). Whereas neither beta-arrestin 1 nor G(alphaq/11) activation alone is sufficient to robustly activate RhoA, the concurrent recruitment of beta-arrestin 1 and activation of G(alphaq/11) leads to full activation of RhoA and to the subsequent formation of stress fibers.

Authors
Barnes, WG; Reiter, E; Violin, JD; Ren, X-R; Milligan, G; Lefkowitz, RJ
MLA Citation
Barnes, WG, Reiter, E, Violin, JD, Ren, X-R, Milligan, G, and Lefkowitz, RJ. "beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation." J Biol Chem 280.9 (March 4, 2005): 8041-8050.
PMID
15611106
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
9
Publish Date
2005
Start Page
8041
End Page
8050
DOI
10.1074/jbc.M412924200

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.

Authors
Tachibana, H; Naga Prasad, SV; Lefkowitz, RJ; Koch, WJ; Rockman, HA
MLA Citation
Tachibana, H, Naga Prasad, SV, Lefkowitz, RJ, Koch, WJ, and Rockman, HA. "Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure." Circulation 111.5 (February 8, 2005): 591-597.
Website
http://hdl.handle.net/10161/5908
PMID
15668342
Source
pubmed
Published In
Circulation
Volume
111
Issue
5
Publish Date
2005
Start Page
591
End Page
597
DOI
10.1161/01.CIR.0000142291.70954.DF

Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor.

Signaling through beta-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for beta-arrestin binding, we studied the roles of different GRKs in promoting beta-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs- and beta-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and beta-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of beta-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and beta-arrestin recruitment, but yet appeared exclusively to support beta-arrestin 2-mediated ERK activation. GRK2 suppression actually increased beta-arrestin-stimulated ERK activation. These results suggest that beta-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.

Authors
Ren, X-R; Reiter, E; Ahn, S; Kim, J; Chen, W; Lefkowitz, RJ
MLA Citation
Ren, X-R, Reiter, E, Ahn, S, Kim, J, Chen, W, and Lefkowitz, RJ. "Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor." Proc Natl Acad Sci U S A 102.5 (February 1, 2005): 1448-1453.
PMID
15671180
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
5
Publish Date
2005
Start Page
1448
End Page
1453
DOI
10.1073/pnas.0409534102

Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling.

beta-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating beta-arrestin-mediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, beta-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2/3. In contrast, inhibiting GRK 5 or 6 expression abolishes beta-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, beta-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of beta-arrestins bound to receptors phosphorylated by different classes of GRKs.

Authors
Kim, J; Ahn, S; Ren, X-R; Whalen, EJ; Reiter, E; Wei, H; Lefkowitz, RJ
MLA Citation
Kim, J, Ahn, S, Ren, X-R, Whalen, EJ, Reiter, E, Wei, H, and Lefkowitz, RJ. "Functional antagonism of different G protein-coupled receptor kinases for beta-arrestin-mediated angiotensin II receptor signaling." Proc Natl Acad Sci U S A 102.5 (February 1, 2005): 1442-1447.
PMID
15671181
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
5
Publish Date
2005
Start Page
1442
End Page
1447
DOI
10.1073/pnas.0409532102

Activation dependent conformational changes in beta-arrestin 2

Authors
Xiao, KH; Shenoy, SK; Nobles, K; Oas, TG; Lefkowitz, RJ
MLA Citation
Xiao, KH, Shenoy, SK, Nobles, K, Oas, TG, and Lefkowitz, RJ. "Activation dependent conformational changes in beta-arrestin 2." January 2005.
Source
wos-lite
Published In
Biophysical Journal
Volume
88
Issue
1
Publish Date
2005
Start Page
554A
End Page
554A

Summary of Wenner-Gren international symposium receptor-receptor interactions among heptaspanning membrane receptors: from structure to function.

Listening to the talks delivered at the symposium during this 2 1/2-d meeting left me with the feeling that this was a field that has truly come of age in the past few years. Although the earliest observations, a number of them by the organizers of the meeting, on receptor-receptor interactions go back almost two decades, it is really only in the past few years that the molecular basis of these interactions has begun to be clarified. The initial skepticism is now subsiding as more and more examples of receptor oligomerization are identified. At the meeting, three types of receptor oligomerization were discussed: homo-oligomerization; hetero-oligomerization, where both partners are seven transmembrane (7TM)-spanning receptors; and hetero-oligomerization, where a 7 TM-spanning receptor is complexed with a structurally different class of molecule. Although interesting papers were presented in each area, I found myself most intrigued by the papers dealing with hetero-oligomerization between different 7TM-spanning receptors. Most striking were examples where novel functions were created by the heterodimerization process, such as novel opioid binding patterns or the acquisition of novel signaling pathways, as in the case of D1-D2 dopamine receptor heterodimerization. In this connection, to me, one of the most interesting discussions concerned the possibility that a number of currently so-called orphan receptors might have no personal ligands but, rather, function to bestow novel properties on other 7TM-spanning receptors by oligomer formation. Thus, these orphans might convey a new signaling function or an altered ligand- binding specificity on a known receptor. If this were the case then, for such an orphan, all efforts to identify its function by expressing it alone would be doomed to fail. Another potentially interesting function of some orphan receptors might be to act as chaperones for other receptors, helping to bring them to the cell surface and stabilizing their membrane expression.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Summary of Wenner-Gren international symposium receptor-receptor interactions among heptaspanning membrane receptors: from structure to function." J Mol Neurosci 26.2-3 (2005): 293-294.
PMID
16012202
Source
pubmed
Published In
Journal of Molecular Neuroscience
Volume
26
Issue
2-3
Publish Date
2005
Start Page
293
End Page
294
DOI
10.1385/JMN:26:2-3:293

The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient ouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation. © 2005 Biochemical Society.

Authors
Milasta, S; Evans, NA; Ormiston, L; Wilson, S; Lefkowitz, RJ; Milligan, G
MLA Citation
Milasta, S, Evans, NA, Ormiston, L, Wilson, S, Lefkowitz, RJ, and Milligan, G. "The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation." Biochemical Journal 387.3 (2005): 573-584.
PMID
15683363
Source
scival
Published In
Biochemical Journal
Volume
387
Issue
3
Publish Date
2005
Start Page
573
End Page
584
DOI
10.1042/BJ20041745

Summary of Wenner-Gren international symposium receptor-receptor interactions among heptaspanning membrane receptors: From structure to function

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Summary of Wenner-Gren international symposium receptor-receptor interactions among heptaspanning membrane receptors: From structure to function." JOURNAL OF MOLECULAR NEUROSCIENCE 26.2-3 (2005): 293-294.
Source
wos-lite
Published In
Journal of Molecular Neuroscience
Volume
26
Issue
2-3
Publish Date
2005
Start Page
293
End Page
294
DOI
10.1385/JMN/26:02:293

Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2.

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.

Authors
Ge, L; Shenoy, SK; Lefkowitz, RJ; DeFea, K
MLA Citation
Ge, L, Shenoy, SK, Lefkowitz, RJ, and DeFea, K. "Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2." J Biol Chem 279.53 (December 31, 2004): 55419-55424.
PMID
15489220
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
53
Publish Date
2004
Start Page
55419
End Page
55424
DOI
10.1074/jbc.M410312200

Activation-dependent conformational changes in {beta}-arrestin 2.

Beta-arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.

Authors
Xiao, K; Shenoy, SK; Nobles, K; Lefkowitz, RJ
MLA Citation
Xiao, K, Shenoy, SK, Nobles, K, and Lefkowitz, RJ. "Activation-dependent conformational changes in {beta}-arrestin 2." J Biol Chem 279.53 (December 31, 2004): 55744-55753.
PMID
15501822
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
53
Publish Date
2004
Start Page
55744
End Page
55753
DOI
10.1074/jbc.M409785200

Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2.

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

Authors
Chen, W; Ren, X-R; Nelson, CD; Barak, LS; Chen, JK; Beachy, PA; de Sauvage, F; Lefkowitz, RJ
MLA Citation
Chen, W, Ren, X-R, Nelson, CD, Barak, LS, Chen, JK, Beachy, PA, de Sauvage, F, and Lefkowitz, RJ. "Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2." Science 306.5705 (December 24, 2004): 2257-2260.
PMID
15618519
Source
pubmed
Published In
Science
Volume
306
Issue
5705
Publish Date
2004
Start Page
2257
End Page
2260
DOI
10.1126/science.1104135

Resonating to the music of ubiquitination.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Resonating to the music of ubiquitination." Nat Methods 1.3 (December 2004): 191-193.
PMID
15782194
Source
pubmed
Published In
Nature Methods
Volume
1
Issue
3
Publish Date
2004
Start Page
191
End Page
193
DOI
10.1038/nmeth1204-191

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching.

Recently, it has been shown that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G(s) and increases its affinity for G(i). Here we demonstrate that, like the beta(2)-AR, the beta(1)-AR is also capable of "switching" its coupling from G(s) to G(i) in a PKA-dependent manner. The beta(1)-AR is capable of activating adenylate cyclase via G(s), and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed beta(1)-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G(i)/G(o), and to the PKA inhibitor, H-89. beta(1)-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G(s)-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the beta(1)-AR, like the beta(2)-AR, can undergo PKA-dependent "G(s)/G(i) switching".

Authors
Martin, NP; Whalen, EJ; Zamah, MA; Pierce, KL; Lefkowitz, RJ
MLA Citation
Martin, NP, Whalen, EJ, Zamah, MA, Pierce, KL, and Lefkowitz, RJ. "PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching." Cell Signal 16.12 (December 2004): 1397-1403.
PMID
15381255
Source
pubmed
Published In
Cellular Signalling
Volume
16
Issue
12
Publish Date
2004
Start Page
1397
End Page
1403
DOI
10.1016/j.cellsig.2004.05.002

Stable interaction between beta-arrestin 2 and angiotensin type 1A receptor is required for beta-arrestin 2-mediated activation of extracellular signal-regulated kinases 1 and 2.

Binding of beta-arrestins to seven-membrane-spanning receptors (7MSRs) not only leads to receptor desensitization and endocytosis but also elicits additional signaling processes. We recently proposed that stimulation of the angiotensin type 1A (AT(1A)) receptor results in independent beta-arrestin 2- and G protein-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation. Here we utilize two AT(1A) mutant receptors to study these independent pathways, one truncated at residue 324, thus removing all potential carboxyl-terminal phosphorylation sites, and the other bearing four mutations in the serine/threonine-rich clusters in the carboxyl terminus. As assessed by confocal microscopy, the two mutant receptors interacted with beta-arrestin 2-green fluorescent protein with much lower affinity than did the wild-type receptor. In addition, the mutant receptors more robustly stimulated G protein-mediated inositol phosphate production. Approximately one-half of the wild-type AT(1A) receptor-stimulated ERK1/2 activation was via a beta-arrestin 2-dependent pathway (suppressed by beta-arrestin 2 small interfering RNA), whereas the rest was mediated by a G protein-dependent pathway (suppressed by protein kinase C inhibitor). ERK1/2 activation by the mutant receptors was insensitive to beta-arrestin 2 small interfering RNA but was reduced more than 80% by a protein kinase C inhibitor. The biochemical consequences of ERK activation by the G protein and beta-arrestin 2-dependent pathways were also distinct. G-protein-mediated ERK activation enhanced the transcription of early growth response 1, whereas beta-arrestin 2-dependent ERK activation did not. In addition, stimulation of the truncated AT(1A) mutant receptor caused significantly greater early growth response 1 transcription than did the wild-type receptor. These findings demonstrate how the ability of receptors to interact with beta-arrestins determines both the mechanism of ERK activation as well as the physiological consequences of this activation.

Authors
Wei, H; Ahn, S; Barnes, WG; Lefkowitz, RJ
MLA Citation
Wei, H, Ahn, S, Barnes, WG, and Lefkowitz, RJ. "Stable interaction between beta-arrestin 2 and angiotensin type 1A receptor is required for beta-arrestin 2-mediated activation of extracellular signal-regulated kinases 1 and 2." J Biol Chem 279.46 (November 12, 2004): 48255-48261.
PMID
15355986
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
46
Publish Date
2004
Start Page
48255
End Page
48261
DOI
10.1074/jbc.M406205200

Use-dependent beta 2-adrenergic receptor-GI coupling requires RGS and beta-ARK/PKA-mediated receptor phosphorylation

Authors
Chakir, K; Zhang, SJ; Rhee, MH; Blumer, K; Lefkowitz, RJ; Xiao, RP
MLA Citation
Chakir, K, Zhang, SJ, Rhee, MH, Blumer, K, Lefkowitz, RJ, and Xiao, RP. "Use-dependent beta 2-adrenergic receptor-GI coupling requires RGS and beta-ARK/PKA-mediated receptor phosphorylation." October 26, 2004.
Source
wos-lite
Published In
Circulation
Volume
110
Issue
17
Publish Date
2004
Start Page
92
End Page
92

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

Authors
Usui, I; Imamura, T; Huang, J; Satoh, H; Shenoy, SK; Lefkowitz, RJ; Hupfeld, CJ; Olefsky, JM
MLA Citation
Usui, I, Imamura, T, Huang, J, Satoh, H, Shenoy, SK, Lefkowitz, RJ, Hupfeld, CJ, and Olefsky, JM. "beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1." Mol Cell Biol 24.20 (October 2004): 8929-8937.
Website
http://hdl.handle.net/10161/7794
PMID
15456867
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
24
Issue
20
Publish Date
2004
Start Page
8929
End Page
8937
DOI
10.1128/MCB.24.20.8929-8937.2004

Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor.

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely G(q)/G(11)) or beta-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized beta-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t((1/2)) approximately 2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, beta-arrestin2-dependent activation was slower (peak 5-10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via beta-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and beta-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.

Authors
Ahn, S; Shenoy, SK; Wei, H; Lefkowitz, RJ
MLA Citation
Ahn, S, Shenoy, SK, Wei, H, and Lefkowitz, RJ. "Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor." J Biol Chem 279.34 (August 20, 2004): 35518-35525.
PMID
15205453
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
34
Publish Date
2004
Start Page
35518
End Page
35525
DOI
10.1074/jbc.M405878200

Historical review: a brief history and personal retrospective of seven-transmembrane receptors.

Pharmacologists have studied receptors for more than a century but a molecular understanding of their properties has emerged only during the past 30-35 years. In this article, I provide a personal retrospective of how developments and discoveries primarily during the 1970s and 1980s led to current concepts about the largest group of receptors, the superfamily of seven-transmembrane (7TM) receptors [also known as G-protein-coupled receptors (GPCRs)]. Significant technical advances such as the development of methods for radioligand binding, solubilization and purification of the beta(2)-adrenoceptor and other adrenoceptors led to the cloning of receptor genes and the discovery of their 7TM architecture and homology with rhodopsin. A universal mechanism of receptor regulation by G-protein-coupled receptor kinases (GRKs) and arrestins, originally discovered as a means of "desensitizing" G-protein-mediated second-messenger generation, was subsequently found to mediate both receptor endocytosis and activation of a growing list of signaling pathways such as those involving mitogen-activated protein kinases. Numerous opportunities for novel therapeutics should emerge from current and future research on 7TM receptor biology.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "Historical review: a brief history and personal retrospective of seven-transmembrane receptors." Trends Pharmacol Sci 25.8 (August 2004): 413-422.
PMID
15276710
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
25
Issue
8
Publish Date
2004
Start Page
413
End Page
422
DOI
10.1016/j.tips.2004.06.006

Relative opioid efficacy is determined by the complements of the G protein-coupled receptor desensitization machinery.

G protein-coupled receptor regulation by G protein-coupled receptor kinases and beta-arrestins can lead to desensitization and subsequent internalization of the receptor. In in vitro and cellular systems, beta-arrestins do not seem to play a major role in regulating micro opioid receptor (microOR) responsiveness. Removal of the betaarrestin2 (betaarr2) gene in mice leads paradoxically to enhanced and prolonged microOR-mediated antinociception. The betaarr2 knockout (betaarr2-KO) mice also fail to develop morphine antinociceptive tolerance in the hot-plate test, further indicating that the betaarr2 protein plays an essential role in microOR regulation in vivo. In this study, the contribution of betaarr2 to the regulation of the microOR was examined in both human embryonic kidney 293 cells and in betaarr2-KO mice after treatment with several opiate agonists. A green fluorescent protein tagged betaarr2 was used to assess receptor-betaarr2 interactions in living cells. Opiate agonists that induced robust betaarr2-green fluorescent protein translocation produced similar analgesia profiles in wild-type and betaarr2-KO mice, whereas those that do not promote robust betaarr2 recruitment, such as morphine and heroin, produce enhanced analgesia in vivo. In this report, we present a rationale to explain the seemingly paradoxical relationship between beta-arrestins and microOR regulation wherein morphine-like agonists fail to promote efficient internalization and resensitization of the receptor.

Authors
Bohn, LM; Dykstra, LA; Lefkowitz, RJ; Caron, MG; Barak, LS
MLA Citation
Bohn, LM, Dykstra, LA, Lefkowitz, RJ, Caron, MG, and Barak, LS. "Relative opioid efficacy is determined by the complements of the G protein-coupled receptor desensitization machinery." Mol Pharmacol 66.1 (July 2004): 106-112.
PMID
15213301
Source
pubmed
Published In
Molecular pharmacology
Volume
66
Issue
1
Publish Date
2004
Start Page
106
End Page
112
DOI
10.1124/mol.66.1.106

beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha.

In addition to their roles in desensitization and signaling of seven-membrane-spanning receptors, beta-arrestins have been more recently implicated in regulating non-seven-membrane-spanning receptor pathways. By using a yeast two-hybrid screen, we identified the inhibitor of NF-kappaB, IkappaBalpha, as a binding partner of beta-arrestin 1. Both beta-arrestin 1 and 2 interact with IkappaBalpha in transfected cells as assessed by immunoprecipitation experiments. Additionally, upstream kinases known to regulate the function of IkappaBalpha, such as IkappaB kinase alpha and beta and NF-kappaB-inducing kinase, were also shown to interact with beta-arrestin. Overexpression of either beta-arrestin 1 or beta-arrestin 2 led to marked inhibition of NF-kappaB activity, as measured by reporter gene activity. Inhibition of NF-kappaB activity was independent of the type of stimulus used for NF-kappaB activation. Conversely, suppression of beta-arrestin 1, but not beta-arrestin 2, expression by using RNA interference led to a 3-fold increase in tumor necrosis factor-stimulated NF-kappaB activity as measured by NF-kappaB mobility-shift analysis. These data uncover a role of beta-arrestins in the regulation of NF-kappaB-mediated gene regulation.

Authors
Witherow, DS; Garrison, TR; Miller, WE; Lefkowitz, RJ
MLA Citation
Witherow, DS, Garrison, TR, Miller, WE, and Lefkowitz, RJ. "beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha." Proc Natl Acad Sci U S A 101.23 (June 8, 2004): 8603-8607.
PMID
15173580
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
101
Issue
23
Publish Date
2004
Start Page
8603
End Page
8607
DOI
10.1073/pnas.0402851101

Anti-β1-adrenergic receptor antibodies and heart failure: Causation, not just correlation

Antibodies specific for the β1-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI (see the related article beginning on page 1419), we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Authors
Freedman, NJ; Lefkowitz, RJ
MLA Citation
Freedman, NJ, and Lefkowitz, RJ. "Anti-β1-adrenergic receptor antibodies and heart failure: Causation, not just correlation." Journal of Clinical Investigation 113.10 (May 1, 2004): 1379-1382. (Review)
Source
scopus
Published In
Journal of Clinical Investigation
Volume
113
Issue
10
Publish Date
2004
Start Page
1379
End Page
1382
DOI
10.1172/JCI200421748

Anti-beta(1)-adrenergic receptor antibodies and heart failure: causation, not just correlation.

Antibodies specific for the beta(1)-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI, we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Authors
Freedman, NJ; Lefkowitz, RJ
MLA Citation
Freedman, NJ, and Lefkowitz, RJ. "Anti-beta(1)-adrenergic receptor antibodies and heart failure: causation, not just correlation." J Clin Invest 113.10 (May 2004): 1379-1382. (Review)
Website
http://hdl.handle.net/10161/5930
PMID
15146232
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
113
Issue
10
Publish Date
2004
Start Page
1379
End Page
1382
DOI
10.1172/JCI21748

beta-arrestins: traffic cops of cell signaling.

Once thought to function only in the desensitization of seven membrane spanning receptors (7MSRs), the ubiquitous beta-arrestin molecules are increasingly appreciated to play important roles in the endocytosis and signaling of these receptors. These functions reflect the ability of the beta-arrestins to bind an ever-growing list of signaling and endocytic elements, often in an agonist-dependent fashion. One heavily studied system is that leading to MAP kinase activation via beta-arrestin-mediated scaffolding of these pathways in a receptor-dependent fashion. The beta-arrestins are also found to be involved in the regulation of novel receptor systems, such as Frizzled and TGFbeta receptors.

Authors
Lefkowitz, RJ; Whalen, EJ
MLA Citation
Lefkowitz, RJ, and Whalen, EJ. "beta-arrestins: traffic cops of cell signaling." Curr Opin Cell Biol 16.2 (April 2004): 162-168. (Review)
PMID
15196559
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
16
Issue
2
Publish Date
2004
Start Page
162
End Page
168
DOI
10.1016/j.ceb.2004.01.001

beta-arrestin-2 regulation of lung eosinophilia in a mouse model of Th2-type airway inflammation

Authors
Walker, JKL; Lawson, BL; Schwartz, DA; Lefkowitz, RJ
MLA Citation
Walker, JKL, Lawson, BL, Schwartz, DA, and Lefkowitz, RJ. "beta-arrestin-2 regulation of lung eosinophilia in a mouse model of Th2-type airway inflammation." March 23, 2004.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
18
Issue
4
Publish Date
2004
Start Page
A463
End Page
A463

Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2.

beta-Arrestin2 not only plays essential roles in seven membrane-spanning receptor desensitization and internalization but also functions as a signal transducer in mitogen-activated protein kinase cascades. Here we show that the angiotensin II type 1A receptor-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HEK-293 cells is increased when the cellular level of beta-arrestin1 is down-regulated by RNA interference but is decreased or eliminated when the cellular level of beta-arrestin2 is diminished. Such reciprocal effects of down-regulated levels of beta-arrestins 1 and 2 are primarily due to differences in the ability of the two forms of beta-arrestins to directly mediate ERK activation. These results are the first to demonstrate reciprocal activity of beta-arrestin isoforms on a signaling pathway and suggest that physiological levels of beta-arrestin1 may act as "dominant-negative" inhibitors of beta-arrestin2-mediated ERK activation.

Authors
Ahn, S; Wei, H; Garrison, TR; Lefkowitz, RJ
MLA Citation
Ahn, S, Wei, H, Garrison, TR, and Lefkowitz, RJ. "Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2." J Biol Chem 279.9 (February 27, 2004): 7807-7811.
PMID
14711824
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
9
Publish Date
2004
Start Page
7807
End Page
7811
DOI
10.1074/jbc.C300443200

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation.

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

Authors
Walker, JKL; Gainetdinov, RR; Feldman, DS; McFawn, PK; Caron, MG; Lefkowitz, RJ; Premont, RT; Fisher, JT
MLA Citation
Walker, JKL, Gainetdinov, RR, Feldman, DS, McFawn, PK, Caron, MG, Lefkowitz, RJ, Premont, RT, and Fisher, JT. "G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation." Am J Physiol Lung Cell Mol Physiol 286.2 (February 2004): L312-L319.
PMID
14565944
Source
pubmed
Published In
American journal of physiology. Lung cellular and molecular physiology
Volume
286
Issue
2
Publish Date
2004
Start Page
L312
End Page
L319
DOI
10.1152/ajplung.00255.2003

Desensitization of G protein-coupled receptors and neuronal functions.

G protein-coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology. Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions. GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein-coupled receptor kinases (GRKs). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and beta arrestins in major brain functions. In particular, screening of genetically modified mice lacking individual GRKs or beta arrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and mu-opioid receptor regulation of locomotion and analgesia. An important and specific role of GRKs and beta arrestins in regulating physiological responsiveness to psychostimulants and morphine suggests potential involvement of these molecules in certain brain disorders, such as addiction, Parkinson's disease, mood disorders, and schizophrenia. Furthermore, the utility of a pharmacological strategy aimed at targeting this GPCR desensitization machinery to regulate brain functions can be envisaged.

Authors
Gainetdinov, RR; Premont, RT; Bohn, LM; Lefkowitz, RJ; Caron, MG
MLA Citation
Gainetdinov, RR, Premont, RT, Bohn, LM, Lefkowitz, RJ, and Caron, MG. "Desensitization of G protein-coupled receptors and neuronal functions." Annu Rev Neurosci 27 (2004): 107-144. (Review)
PMID
15217328
Source
pubmed
Published In
Annual Review of Neuroscience
Volume
27
Publish Date
2004
Start Page
107
End Page
144
DOI
10.1146/annurev.neuro.27.070203.144206

Neuropathic pain activates the endogenous κ opioid system in mouse spinal cord and induces opioid receptor tolerance

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [κ opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the κ agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Authors
Xu, M; Petraschka, M; McLaughlin, JP; Westenbroek, RE; Caron, MG; Lefkowitz, RJ; Czyzyk, TA; Pintar, JE; Terman, GW; Chavkin, C
MLA Citation
Xu, M, Petraschka, M, McLaughlin, JP, Westenbroek, RE, Caron, MG, Lefkowitz, RJ, Czyzyk, TA, Pintar, JE, Terman, GW, and Chavkin, C. "Neuropathic pain activates the endogenous κ opioid system in mouse spinal cord and induces opioid receptor tolerance." Journal of Neuroscience 24.19 (2004): 4576-4584.
Website
http://hdl.handle.net/10161/5936
PMID
15140929
Source
scival
Published In
Journal of Neuroscience
Volume
24
Issue
19
Publish Date
2004
Start Page
4576
End Page
4584
DOI
10.1523/JNEUROSCI.5552-03.2004

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2 muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5-/-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5-/- and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from β2-adrenergic receptor activation were diminished in GRK5-/- mice. These data suggest that M2 muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5-/- mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

Authors
Walker, JKL; Gainetdinov, RR; Feldman, DS; McFawn, PK; Caron, MG; Lefkowitz, RJ; Premont, RT; Fisher, JT
MLA Citation
Walker, JKL, Gainetdinov, RR, Feldman, DS, McFawn, PK, Caron, MG, Lefkowitz, RJ, Premont, RT, and Fisher, JT. "G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation." American Journal of Physiology - Lung Cellular and Molecular Physiology 286.2 30-2 (2004): L312-L319.
Source
scival
Published In
American Journal of Physiology - Lung Cellular and Molecular Physiology
Volume
286
Issue
2 30-2
Publish Date
2004
Start Page
L312
End Page
L319

Prolonged Kappa Opioid Receptor Phosphorylation Mediated by G-protein Receptor Kinase Underlies Sustained Analgesic Tolerance

Kappa opioid receptor (KOR) desensitization was previously shown to follow agonist-dependent phosphorylation of serine 369 by G-protein receptor kinase (GRK) and β-arrestin binding in transfected cells. To study the in vivo effects induced by phosphorylation of KOR(S369), C57B1/6 mice were administered single or repeated doses of the KOR agonist, U50,488, and isolated brain glycoprotein was probed with an antibody, KOR-P, that specifically recognized phosphoserine 369 KOR. Western blot analysis using KOR-P antibody showed that labeling intensity increased after either single or repeated treatment of mice with U50,488 by 59 ± 22% and 101 ± 29%, respectively. In contrast, there was no change in labeling intensity by nonphosphoselective KOR antibodies following acute or chronic in vivo treatment with kappa agonist. Moreover, mice lacking GRK3 showed no increase in KOR-P labeling and developed significantly less analgesic tolerance following treatment with kappa agonist. The result suggests that tolerance to kappa agonists includes phosphorylation of serine 369 within KOR by GRK3. Recovery of analgesic potency and reduction of elevated KOR-P labeling in wild-type mice both required 2 weeks to return to base line. Consistent with these results, in vitro phosphorylation by GRK3 of KOR isolated from tolerant mice resulted in 46 ± 7% less 32P incorporation than in KOR isolated from untreated mice. In addition, in vitro 32P incorporation returned to base line levels only in KOR isolated from tolerant mice allowed to recover for 2 weeks. The coincident reversal of analgesic tolerance and slow return to a basal phosphorylation state matched the regeneration rate of functional kappa receptors following irreversible antagonism and suggested that receptor replacement rather than dephosphorylation was required to restore sensitivity.

Authors
McLaughlin, JP; Myers, LC; Zarek, PE; Caron, MG; Lefkowitz, RJ; Czyzyk, TA; Pintar, JE; Chavkin, C
MLA Citation
McLaughlin, JP, Myers, LC, Zarek, PE, Caron, MG, Lefkowitz, RJ, Czyzyk, TA, Pintar, JE, and Chavkin, C. "Prolonged Kappa Opioid Receptor Phosphorylation Mediated by G-protein Receptor Kinase Underlies Sustained Analgesic Tolerance." Journal of Biological Chemistry 279.3 (2004): 1810-1818.
PMID
14597630
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
3
Publish Date
2004
Start Page
1810
End Page
1818
DOI
10.1074/jbc.M305796200

G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal

1. Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G-protein receptor kinase mediated receptor phosphorylation and subsequent β-arrestin binding. To define the role of this regulatory mechanism in the control of the electrophysiological and behavioral responses to opioids, we used mice having a targeted disruption of the G-protein receptor kinase 3 (GRK3) gene. 2. Mice lacking GRK3 did not differ from wild-type littermates neither in their response latencies to noxious stimuli on the hot-plate test nor in their acute antinociceptive responses to fentanyl or morphine. 3. Tolerance to the electrophysiological response to the opioid fentanyl, measured in vitro in the hippocampus, was blocked by GRK3 deletion. In addition, tolerance to the antinociceptive effects of fentanyl was significantly reduced in GRK3 knockouts compared to wild-type littermate controls. 4. Tolerance to the antinociceptive effects of morphine was not affected by GRK3 deletion although morphine tolerance in hippocampal slices from GRK3 knockout mice was significantly inhibited. Tolerance developed more slowly in vitro to morphine than fentanyl supporting previous work in in vitro systems showing a correlation between agonist efficacy and GRK3-mediated desensitization. 5. The results of these studies suggest that GRK3-mediated mechanisms are important components of both electrophysiologic and behavioral opioid tolerance. Fentanyl, a high efficacy opioid, more effectively produced GRK3-dependent effects than morphine, a low efficacy agonist.

Authors
Terman, GW; Jin, W; Cheong, Y-P; Lowe, J; Caron, MG; Lefkowitz, RJ; Chavkin, C
MLA Citation
Terman, GW, Jin, W, Cheong, Y-P, Lowe, J, Caron, MG, Lefkowitz, RJ, and Chavkin, C. "G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal." British Journal of Pharmacology 141.1 (2004): 55-64.
PMID
14662727
Source
scival
Published In
British Journal of Pharmacology
Volume
141
Issue
1
Publish Date
2004
Start Page
55
End Page
64
DOI
10.1038/sj.bjp.0705595

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca2+ imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Gα subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.

Authors
Fukuto, HS; Ferkey, DM; Apicella, AJ; Lans, H; Sharmeen, T; Chen, W; Lefkowitz, RJ; Jansen, G; Schafer, WR; Hart, AC
MLA Citation
Fukuto, HS, Ferkey, DM, Apicella, AJ, Lans, H, Sharmeen, T, Chen, W, Lefkowitz, RJ, Jansen, G, Schafer, WR, and Hart, AC. "G protein-coupled receptor kinase function is essential for chemosensation in C. elegans." Neuron 42.4 (2004): 581-593.
PMID
15157420
Source
scival
Published In
Neuron
Volume
42
Issue
4
Publish Date
2004
Start Page
581
End Page
593
DOI
10.1016/S0896-6273(04)00252-1

Anti-β1-adrenergic receptor antibodies and heart failure: Causation, not just correlation

Antibodies specific for the β1-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI (see the related article beginning on page 1419), we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Authors
Freedman, NJ; Lefkowitz, RJ
MLA Citation
Freedman, NJ, and Lefkowitz, RJ. "Anti-β1-adrenergic receptor antibodies and heart failure: Causation, not just correlation." Journal of Clinical Investigation 113.10 (2004): 1379-1382.
Website
http://hdl.handle.net/10161/7795
Source
scival
Published In
Journal of Clinical Investigation
Volume
113
Issue
10
Publish Date
2004
Start Page
1379
End Page
1382
DOI
10.1172/JCI200421748

The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels

Epithelial Na+ channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel β subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na+ channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

Authors
Dinudom, A; Fotia, AB; Lefkowitz, RJ; Young, JA; Kumar, S; Cook, DI
MLA Citation
Dinudom, A, Fotia, AB, Lefkowitz, RJ, Young, JA, Kumar, S, and Cook, DI. "The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels." Proceedings of the National Academy of Sciences of the United States of America 101.32 (2004): 11886-11890.
Website
http://hdl.handle.net/10161/7796
PMID
15284439
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
101
Issue
32
Publish Date
2004
Start Page
11886
End Page
11890
DOI
10.1073/pnas.0402178101

Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors

Arrestin regulates almost all G protein-coupled receptor (GPCR)-mediated signaling and trafficking, We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gβγ complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of α2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.

Authors
Wang, Q; Zhao, J; Brady, AE; Feng, J; Allon, PB; Lefkowitz, RJ; Greengard, P; Limbird, LE
MLA Citation
Wang, Q, Zhao, J, Brady, AE, Feng, J, Allon, PB, Lefkowitz, RJ, Greengard, P, and Limbird, LE. "Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors." Science 304.5679 (2004): 1940-1944.
PMID
15218143
Source
scival
Published In
Science
Volume
304
Issue
5679
Publish Date
2004
Start Page
1940
End Page
1944
DOI
10.1126/science.1098274

β-arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6

Seven transmembrane receptors mediate diverse physiological responses including hormone action, o1-faction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor β-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with β-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from β-arrestin activate any G-protein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.

Authors
Galliera, E; Jala, VR; Trent, JO; Bonecchi, R; Signorelli, P; Lefkowitz, RJ; Mantovani, A; Locati, M; Haribabu, B
MLA Citation
Galliera, E, Jala, VR, Trent, JO, Bonecchi, R, Signorelli, P, Lefkowitz, RJ, Mantovani, A, Locati, M, and Haribabu, B. "β-arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6." Journal of Biological Chemistry 279.24 (2004): 25590-25597.
PMID
15084596
Source
scival
Published In
Journal of Biological Chemistry
Volume
279
Issue
24
Publish Date
2004
Start Page
25590
End Page
25597
DOI
10.1074/jbc.M400363200

beta-arrestins: Master regulators of seven membrane spanning receptors

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "beta-arrestins: Master regulators of seven membrane spanning receptors." 2004.
Source
wos-lite
Published In
Journal of pharmacological sciences
Volume
94
Publish Date
2004
Start Page
2P
End Page
2P

Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis.

beta-arrestins (1 and 2) are widely expressed cytosolic proteins that play central roles in G protein-coupled receptor signaling. beta-arrestin1 is also recruited to the insulin-like growth factor 1 (IGF-1) receptor, a receptor tyrosine kinase, upon agonist binding. Here we report that, in response to IGF-1 stimulation, beta-arrestin1 mediates activation of phosphatidylinositol 3-kinase in a pathway that leads to the subsequent activation of Akt and anti-apoptosis. This process is independent of both Gi and ERK activity. The pathway fails in mouse embryo fibroblasts lacking both beta-arrestins and is restored by stable transfection of beta-arrestin1. Remarkably, this pathway is insensitive to chemical inhibition of IGF-1 receptor tyrosine kinase activity. These results suggest that, in addition to their roles in G protein-coupled receptor signaling, beta-arrestins couple the IGF-1 receptor tyrosine kinase to the phosphatidylinositol 3-kinase system and suggest that this mechanism is operative independently of the tyrosine kinase activity of the receptor.

Authors
Povsic, TJ; Kohout, TA; Lefkowitz, RJ
MLA Citation
Povsic, TJ, Kohout, TA, and Lefkowitz, RJ. "Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis." J Biol Chem 278.51 (December 19, 2003): 51334-51339.
PMID
14534298
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
51
Publish Date
2003
Start Page
51334
End Page
51339
DOI
10.1074/jbc.M309968200

Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6.

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.

Authors
Kavelaars, A; Vroon, A; Raatgever, RP; Fong, AM; Premont, RT; Patel, DD; Lefkowitz, RJ; Heijnen, CJ
MLA Citation
Kavelaars, A, Vroon, A, Raatgever, RP, Fong, AM, Premont, RT, Patel, DD, Lefkowitz, RJ, and Heijnen, CJ. "Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6." J Immunol 171.11 (December 1, 2003): 6128-6134.
PMID
14634128
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
171
Issue
11
Publish Date
2003
Start Page
6128
End Page
6134

Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination.

The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R. A mutant V2R (K268R) that is not ubiquitinated still activates Gs and internalizes with similar kinetics as the wild type receptor. Unstimulated wild type and K268R mutant receptors degrade at similar rates and have comparable half-lives of 217 +/- 17 and 245 +/- 29 min as determined by pulse-chase experiments. However, following agonist stimulation, the rate of receptor degradation for the wild type is enhanced (half-life of 69 +/- 19 min), whereas that of the mutant is only minimally affected (half-life of 188 +/- 11 min). These data suggest that V2R levels are regulated through at least two processes. In the absence of agonist stimulation, a slow degradative pathway operates that is independent of receptor ubiquitination. However, receptor stimulation leads to rapid beta-arrestin2-dependent ubiquitination of the receptor and increased degradation.

Authors
Martin, NP; Lefkowitz, RJ; Shenoy, SK
MLA Citation
Martin, NP, Lefkowitz, RJ, and Shenoy, SK. "Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination." J Biol Chem 278.46 (November 14, 2003): 45954-45959.
PMID
12960162
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
46
Publish Date
2003
Start Page
45954
End Page
45959
DOI
10.1074/jbc.M308285200

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Authors
Bohn, LM; Gainetdinov, RR; Sotnikova, TD; Medvedev, IO; Lefkowitz, RJ; Dykstra, LA; Caron, MG
MLA Citation
Bohn, LM, Gainetdinov, RR, Sotnikova, TD, Medvedev, IO, Lefkowitz, RJ, Dykstra, LA, and Caron, MG. "Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice." J Neurosci 23.32 (November 12, 2003): 10265-10273.
Website
http://hdl.handle.net/10161/5935
PMID
14614085
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
23
Issue
32
Publish Date
2003
Start Page
10265
End Page
10273

Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.

Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling." Biochem J 375.Pt 3 (November 1, 2003): 503-515. (Review)
PMID
12959637
Source
pubmed
Published In
The Biochemical journal
Volume
375
Issue
Pt 3
Publish Date
2003
Start Page
503
End Page
515
DOI
10.1042/BJ20031076

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

Authors
Wei, H; Ahn, S; Shenoy, SK; Karnik, SS; Hunyady, L; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Wei, H, Ahn, S, Shenoy, SK, Karnik, SS, Hunyady, L, Luttrell, LM, and Lefkowitz, RJ. "Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2." Proc Natl Acad Sci U S A 100.19 (September 16, 2003): 10782-10787.
Website
http://hdl.handle.net/10161/7797
PMID
12949261
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
19
Publish Date
2003
Start Page
10782
End Page
10787
DOI
10.1073/pnas.1834556100

Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways.

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.

Authors
Rapacciuolo, A; Suvarna, S; Barki-Harrington, L; Luttrell, LM; Cong, M; Lefkowitz, RJ; Rockman, HA
MLA Citation
Rapacciuolo, A, Suvarna, S, Barki-Harrington, L, Luttrell, LM, Cong, M, Lefkowitz, RJ, and Rockman, HA. "Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways." J Biol Chem 278.37 (September 12, 2003): 35403-35411.
PMID
12821660
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
37
Publish Date
2003
Start Page
35403
End Page
35411
DOI
10.1074/jbc.M305675200

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling.

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.

Authors
Chen, W; Kirkbride, KC; How, T; Nelson, CD; Mo, J; Frederick, JP; Wang, X-F; Lefkowitz, RJ; Blobe, GC
MLA Citation
Chen, W, Kirkbride, KC, How, T, Nelson, CD, Mo, J, Frederick, JP, Wang, X-F, Lefkowitz, RJ, and Blobe, GC. "Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling." Science 301.5638 (September 5, 2003): 1394-1397.
PMID
12958365
Source
pubmed
Published In
Science
Volume
301
Issue
5638
Publish Date
2003
Start Page
1394
End Page
1397
DOI
10.1126/science.1083195

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.

Authors
Chen, W; ten Berge, D; Brown, J; Ahn, S; Hu, LA; Miller, WE; Caron, MG; Barak, LS; Nusse, R; Lefkowitz, RJ
MLA Citation
Chen, W, ten Berge, D, Brown, J, Ahn, S, Hu, LA, Miller, WE, Caron, MG, Barak, LS, Nusse, R, and Lefkowitz, RJ. "Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4." Science 301.5638 (September 5, 2003): 1391-1394.
PMID
12958364
Source
pubmed
Published In
Science
Volume
301
Issue
5638
Publish Date
2003
Start Page
1391
End Page
1394
DOI
10.1126/science.1082808

Beta-arrestin-2 regulates the development of allergic asthma.

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Authors
Walker, JKL; Fong, AM; Lawson, BL; Savov, JD; Patel, DD; Schwartz, DA; Lefkowitz, RJ
MLA Citation
Walker, JKL, Fong, AM, Lawson, BL, Savov, JD, Patel, DD, Schwartz, DA, and Lefkowitz, RJ. "Beta-arrestin-2 regulates the development of allergic asthma." J Clin Invest 112.4 (August 2003): 566-574.
Website
http://hdl.handle.net/10161/5926
PMID
12925697
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
112
Issue
4
Publish Date
2003
Start Page
566
End Page
574
DOI
10.1172/JCI17265

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

Authors
Hu, LA; Chen, W; Martin, NP; Whalen, EJ; Premont, RT; Lefkowitz, RJ
MLA Citation
Hu, LA, Chen, W, Martin, NP, Whalen, EJ, Premont, RT, and Lefkowitz, RJ. "GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation." J Biol Chem 278.28 (July 11, 2003): 26295-26301.
PMID
12724327
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
28
Publish Date
2003
Start Page
26295
End Page
26301
DOI
10.1074/jbc.M212352200

G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GRKs and subsequent recruitment of beta-arrestins to agonist-occupied receptors serves to terminate or attenuate signaling by blocking G-proteins from further interaction with the receptors. Human cytomegalovirus encodes a GPCR termed US28 that is homologous to the human chemokine family of GPCRs but differs from the cellular receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is constitutively active, mechanisms that regulate this activity are unknown. We provide evidence that US28 is constitutively phosphorylated by GRKs in cells and that in consequence, beta-arrestin 2 is localized to the plasma membrane. Deletion of the carboxyl terminal 40 amino acids in US28 generates a receptor that is severely impaired in its ability to become phosphorylated and recruit beta-arrestin and accordingly demonstrates increased inositol phosphate signaling. This result indicates that the carboxyl terminus of US28 contains an important signaling regulatory region and mutational analysis deleting carboxyl terminal serines identified serine 323 as a critical residue within this region. In addition, overexpression of wild type GRK5 leads to hyperphosphorylation of US28 that results in a decrease of inositol phosphate accumulation. These results are consistent with the hypothesis that GRK phosphorylation and recruitment of beta-arrestin to the US28 viral GPCR attenuates signaling to the traditional Galphaq-stimulated inositol phosphate pathway. Finally, in contrast to the results with inositol phosphate signaling, we provide evidence that the US28 carboxyl-terminal phosphorylation sites and beta-arrestin-interacting domain are required for maximal activation of the p38 mitogen-activated protein kinase. Taken together, these results indicate that US28 interacts with these important regulatory proteins to control multiple aspects of signal transmission. Understanding the regulation of viral GPCRs by GRKs and beta-arrestins will provide important new insights into not only aspects of viral pathogenesis but also basic mechanisms of receptor signaling.

Authors
Miller, WE; Houtz, DA; Nelson, CD; Kolattukudy, PE; Lefkowitz, RJ
MLA Citation
Miller, WE, Houtz, DA, Nelson, CD, Kolattukudy, PE, and Lefkowitz, RJ. "G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR." J Biol Chem 278.24 (June 13, 2003): 21663-21671.
PMID
12668664
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
24
Publish Date
2003
Start Page
21663
End Page
21671
DOI
10.1074/jbc.M303219200

Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice.

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Here we show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity.

Authors
Gainetdinov, RR; Bohn, LM; Sotnikova, TD; Cyr, M; Laakso, A; Macrae, AD; Torres, GE; Kim, KM; Lefkowitz, RJ; Caron, MG; Premont, RT
MLA Citation
Gainetdinov, RR, Bohn, LM, Sotnikova, TD, Cyr, M, Laakso, A, Macrae, AD, Torres, GE, Kim, KM, Lefkowitz, RJ, Caron, MG, and Premont, RT. "Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice." Neuron 38.2 (April 24, 2003): 291-303.
PMID
12718862
Source
pubmed
Published In
Neuron
Volume
38
Issue
2
Publish Date
2003
Start Page
291
End Page
303

Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination.

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta(2)-adrenergic receptor (beta(2)AR) this requires ubiquitination of beta-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. beta(2)AR), which recycle rapidly, beta-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, beta-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the beta(2)AR or V2 vasopressin receptor leads, respectively, to transient or stable beta-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of beta-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (beta(2)AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both beta-arrestin trafficking and beta-arrestin ubiquitination. To explore the functional consequences of beta-arrestin ubiquitination we constructed a yellow fluorescent protein-tagged beta-arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" beta-arrestin did not dissociate from the beta(2)AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this beta-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of beta(2)AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and beta-arrestin was increased dramatically, suggesting that deubiquitination of beta-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of beta-arrestin determines the stability of the receptor-beta-arrestin complex as well as the trafficking pattern of beta-arrestin.

Authors
Shenoy, SK; Lefkowitz, RJ
MLA Citation
Shenoy, SK, and Lefkowitz, RJ. "Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination." J Biol Chem 278.16 (April 18, 2003): 14498-14506.
PMID
12574160
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
16
Publish Date
2003
Start Page
14498
End Page
14506
DOI
10.1074/jbc.M209626200

An interview with Professor Robert J. Lefkowitz, MD

Authors
Glaser, V; Lefkowitz, RJ
MLA Citation
Glaser, V, and Lefkowitz, RJ. "An interview with Professor Robert J. Lefkowitz, MD." ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES 1.2 (April 2003): 233-238.
Website
http://hdl.handle.net/10161/7798
PMID
15090188
Source
wos-lite
Published In
ASSAY and Drug Development Technologies
Volume
1
Issue
2
Publish Date
2003
Start Page
233
End Page
238

[beta]-arrestin-2 regulates the development of allergic asthma in mice

Authors
Walker, JKL; Lawson, BL; Savov, JD; Schwartz, DA; Lefkowitz, RJ
MLA Citation
Walker, JKL, Lawson, BL, Savov, JD, Schwartz, DA, and Lefkowitz, RJ. "[beta]-arrestin-2 regulates the development of allergic asthma in mice." March 17, 2003.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
17
Issue
5
Publish Date
2003
Start Page
A833
End Page
A833

The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation.

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-beta-arrestin complexes, activated a beta-arrestin-bound pool of ERK2 more efficiently than alpha 1b and beta2 adrenergic receptors, which form transient receptor-beta-arrestin complexes. We next studied chimeric receptors in which the pattern of beta-arrestin binding was reversed by exchanging the C-terminal tails of the beta2 and V2 receptors. The ability of the V2 beta 2 and beta 2V2 chimeras to activate beta-arrestin-bound ERK2 corresponded to the pattern of beta-arrestin binding, suggesting that the stability of the receptor-beta-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2 beta 2 receptor, consistent with the cytosolic retention of beta-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2 beta 2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2 beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta-arrestin complex, controls the extent of beta-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.

Authors
Tohgo, A; Choy, EW; Gesty-Palmer, D; Pierce, KL; Laporte, S; Oakley, RH; Caron, MG; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Tohgo, A, Choy, EW, Gesty-Palmer, D, Pierce, KL, Laporte, S, Oakley, RH, Caron, MG, Lefkowitz, RJ, and Luttrell, LM. "The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation." J Biol Chem 278.8 (February 21, 2003): 6258-6267.
PMID
12473660
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
8
Publish Date
2003
Start Page
6258
End Page
6267
DOI
10.1074/jbc.M212231200

Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference.

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the beta-arrestins in many seven-membrane-spanning receptor systems.

Authors
Ahn, S; Nelson, CD; Garrison, TR; Miller, WE; Lefkowitz, RJ
MLA Citation
Ahn, S, Nelson, CD, Garrison, TR, Miller, WE, and Lefkowitz, RJ. "Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference." Proc Natl Acad Sci U S A 100.4 (February 18, 2003): 1740-1744.
Website
http://hdl.handle.net/10161/7800
PMID
12582207
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
4
Publish Date
2003
Start Page
1740
End Page
1744
DOI
10.1073/pnas.262789099

Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization.

Authors
Kohout, TA; Lefkowitz, RJ
MLA Citation
Kohout, TA, and Lefkowitz, RJ. "Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization." Mol Pharmacol 63.1 (January 2003): 9-18. (Review)
PMID
12488531
Source
pubmed
Published In
Molecular pharmacology
Volume
63
Issue
1
Publish Date
2003
Start Page
9
End Page
18

N-Formyl Peptide Receptors Internalize but Do Not Recycle in the Absence of Arrestins

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knock-out cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonsrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the postendocytic trafficking of GPCRs.

Authors
Vines, CM; Revankar, CM; Maestas, DC; LaRusch, LL; Cimino, DF; Kohout, TA; Lefkowitz, RJ; Prossnitz, ER
MLA Citation
Vines, CM, Revankar, CM, Maestas, DC, LaRusch, LL, Cimino, DF, Kohout, TA, Lefkowitz, RJ, and Prossnitz, ER. "N-Formyl Peptide Receptors Internalize but Do Not Recycle in the Absence of Arrestins." Journal of Biological Chemistry 278.43 (2003): 41581-41584.
PMID
12947104
Source
scival
Published In
Journal of Biological Chemistry
Volume
278
Issue
43
Publish Date
2003
Start Page
41581
End Page
41584
DOI
10.1074/jbc.C300291200

β-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking β-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the β-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking β-arrestin-2. β-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that β-arrestin-2 is required for the manifestation of allergic asthma. Because β-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Authors
Walker, JKL; Fong, AM; Lawson, BL; Savov, JD; Patel, DD; Schwartz, DA; Lefkowitz, RJ
MLA Citation
Walker, JKL, Fong, AM, Lawson, BL, Savov, JD, Patel, DD, Schwartz, DA, and Lefkowitz, RJ. "β-Arrestin-2 regulates the development of allergic asthma." Journal of Clinical Investigation 112.4 (2003): 566-574.
Website
http://hdl.handle.net/10161/7802
Source
scival
Published In
Journal of Clinical Investigation
Volume
112
Issue
4
Publish Date
2003
Start Page
566
End Page
574
DOI
10.1172/JCI200317265

Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gβ1γ2

G protein-coupled receptor kinase 2 (GRK2) phosphorylates activated G protein-coupled receptors (GPCRs), which ultimately leads to their desensitization and/or downregulation. The enzyme is recruited to the plasma membrane via the interaction of its carboxyl-terminal pleckstrin-homology (PH) domain with the β and γ subunits of heterotrimeric G proteins (Gβγ). An improved purification scheme for GRK2 has been developed, conditions under which GRK2 forms a complex with Gβ1γ2 have been determined and the complex has been crystallized in CHAPS detergent micelles. Crystals of the GRK2-Gβγ complex belong to space group C2 and have unit-cell parameters a = 187.0, b = 72.1, c = 122.0 Å, β = 115.2°. A complete data set has been collected to 3.2 Å resolution with Cu Kα radiation.

Authors
Lodowski, DT; Barnhill, JF; Pitcher, JA; Capel, WD; Lefkowitz, RJ; Tesmer, JJG
MLA Citation
Lodowski, DT, Barnhill, JF, Pitcher, JA, Capel, WD, Lefkowitz, RJ, and Tesmer, JJG. "Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gβ1γ2." Acta Crystallographica - Section D Biological Crystallography 59.5 (2003): 936-939.
Website
http://hdl.handle.net/10161/7801
PMID
12777817
Source
scival
Published In
Acta Crystallographica - Section D Biological Crystallography
Volume
59
Issue
5
Publish Date
2003
Start Page
936
End Page
939
DOI
10.1107/S0907444903002622

Keeping G proteins at bay: A complex between G protein-coupled receptor kinase 2 and Gβγ

The phosphorylation of heptahelical receptors by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor kinases (GRKs) is a universal regulatory mechanism that leads to desensitization of G protein signaling and to the activation of alternative signaling pathways. We determined the crystallographic structure of bovine GRK2 in complex with G protein β1γ2 subunits. Our results show how the three domains of GRK2 - the RGS (regulator of G protein signaling) homology, protein kinase, and pleckstrin homology domains - integrate their respective activities and recruit the enzyme to the cell membrane in an orientation that not only facilitates receptor phosphorylation, but also allows for the simultaneous inhibition of signaling by Gα and Gβγ subunits.

Authors
Lodowski, DT; Pitcher, JA; Capel, WD; Lefkowitz, RJ; Tesmer, JJG
MLA Citation
Lodowski, DT, Pitcher, JA, Capel, WD, Lefkowitz, RJ, and Tesmer, JJG. "Keeping G proteins at bay: A complex between G protein-coupled receptor kinase 2 and Gβγ." Science 300.5623 (2003): 1256-1262.
PMID
12764189
Source
scival
Published In
Science
Volume
300
Issue
5623
Publish Date
2003
Start Page
1256
End Page
1262
DOI
10.1126/science.1082348

A magnificent time with the "magnificent seven" transmembrane spanning receptors

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "A magnificent time with the "magnificent seven" transmembrane spanning receptors." Circulation Research 92.4 (2003): 342-344.
Website
http://hdl.handle.net/10161/7799
PMID
12623870
Source
scival
Published In
Circulation Research
Volume
92
Issue
4
Publish Date
2003
Start Page
342
End Page
344
DOI
10.1161/01.RES.0000061771.61280.B8

β-arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi

Phosphorylation of the β2 adrenoreceptor (β2AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (Gs) to inhibitory guanine nucleotide regulatory protein (Gi). β-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the β2AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the β2AR. In human embryonic kidney 293 cells overexpressing a recombinant β2AR, stimulation with isoprenaline recruits β-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the β2AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the β2AR. Rolipram and PDE4D5-D556A also enhance β2AR-mediated activation of extracellular signal-regulated kinases ERK1/2. This is consistent with a switch in coupling of the receptor from Gs to Gi, because the ERK1/2 activation is sensitive to both inhibitors of PKA (H89) and Gi (pertussis toxin). In cardiac myocytes, the β2AR also switches from Gs to Gi coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1/2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1/2 activation. Thus, receptor-stimulated β-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the β2AR in a physiological system, the cardiac myocyte.

Authors
Baillie, GS; Sood, A; McPhee, I; Gall, I; Perry, SJ; Lefkowitz, RJ; Houslay, MD
MLA Citation
Baillie, GS, Sood, A, McPhee, I, Gall, I, Perry, SJ, Lefkowitz, RJ, and Houslay, MD. "β-arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi." Proceedings of the National Academy of Sciences of the United States of America 100.3 (2003): 940-945.
Website
http://hdl.handle.net/10161/7803
PMID
12552097
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
100
Issue
3
Publish Date
2003
Start Page
940
End Page
945
DOI
10.1073/pnas.262787199

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Authors
Gilman, AG; Simon, MI; Bourne, HR; Harris, BA; Long, R; Ross, EM; Stull, JT; Taussig, R; Bourne, HR; Arkin, AP; Cobb, MH; Cyster, JG; Devreotes, PN; Ferrell, JE; Fruman, D; Gold, M; Weiss, A; Stull, JT; Berridge, MJ; Cantley, LC; Catterall, WA; Coughlin, SR; Olson, EN; Smith, TF; Brugge, JS; Botstein, D; Dixon, JE; Hunter, T; Lefkowitz, RJ; Pawson, AJ; Sternberg, PW; Varmus, H; Subramaniam, S; Sinkovits, RS; Li, J; Mock, D; Ning, Y; Saunders, B; Sternweis, PC; Hilgemann, D; Scheuermann, RH et al.
MLA Citation
Gilman, AG, Simon, MI, Bourne, HR, Harris, BA, Long, R, Ross, EM, Stull, JT, Taussig, R, Bourne, HR, Arkin, AP, Cobb, MH, Cyster, JG, Devreotes, PN, Ferrell, JE, Fruman, D, Gold, M, Weiss, A, Stull, JT, Berridge, MJ, Cantley, LC, Catterall, WA, Coughlin, SR, Olson, EN, Smith, TF, Brugge, JS, Botstein, D, Dixon, JE, Hunter, T, Lefkowitz, RJ, Pawson, AJ, Sternberg, PW, Varmus, H, Subramaniam, S, Sinkovits, RS, Li, J, Mock, D, Ning, Y, Saunders, B, Sternweis, PC, Hilgemann, D, and Scheuermann, RH et al. "Overview of the Alliance for Cellular Signaling." Nature 420.6916 (December 12, 2002): 703-706.
PMID
12478301
Source
pubmed
Published In
Nature
Volume
420
Issue
6916
Publish Date
2002
Start Page
703
End Page
706
DOI
10.1038/nature01304

Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Authors
Bohn, LM; Lefkowitz, RJ; Caron, MG
MLA Citation
Bohn, LM, Lefkowitz, RJ, and Caron, MG. "Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice." J Neurosci 22.23 (December 1, 2002): 10494-10500.
Website
http://hdl.handle.net/10161/5920
PMID
12451149
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
22
Issue
23
Publish Date
2002
Start Page
10494
End Page
10500

Cardiac overexpression of beta-arrestin-2 in transgenic mice accelerates cardiac dysfunction following chronic pressure overload

Authors
Povsic, TJ; Kohout, TA; Mao, L; Rockman, H; Lefkowitz, RJ
MLA Citation
Povsic, TJ, Kohout, TA, Mao, L, Rockman, H, and Lefkowitz, RJ. "Cardiac overexpression of beta-arrestin-2 in transgenic mice accelerates cardiac dysfunction following chronic pressure overload." November 5, 2002.
Source
wos-lite
Published In
Circulation
Volume
106
Issue
19
Publish Date
2002
Start Page
59
End Page
59

Dancing with different partners: protein kinase a phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity.

Authors
Lefkowitz, RJ; Pierce, KL; Luttrell, LM
MLA Citation
Lefkowitz, RJ, Pierce, KL, and Luttrell, LM. "Dancing with different partners: protein kinase a phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity." Mol Pharmacol 62.5 (November 2002): 971-974.
PMID
12391258
Source
pubmed
Published In
Molecular pharmacology
Volume
62
Issue
5
Publish Date
2002
Start Page
971
End Page
974

Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins.

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.

Authors
Perry, SJ; Baillie, GS; Kohout, TA; McPhee, I; Magiera, MM; Ang, KL; Miller, WE; McLean, AJ; Conti, M; Houslay, MD; Lefkowitz, RJ
MLA Citation
Perry, SJ, Baillie, GS, Kohout, TA, McPhee, I, Magiera, MM, Ang, KL, Miller, WE, McLean, AJ, Conti, M, Houslay, MD, and Lefkowitz, RJ. "Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins." Science 298.5594 (October 25, 2002): 834-836.
PMID
12399592
Source
pubmed
Published In
Science
Volume
298
Issue
5594
Publish Date
2002
Start Page
834
End Page
836
DOI
10.1126/science.1074683

Beta 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2.

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

Authors
Freeman, JLR; Gonzalo, P; Pitcher, JA; Claing, A; Lavergne, J-P; Reboud, J-P; Lefkowitz, RJ
MLA Citation
Freeman, JLR, Gonzalo, P, Pitcher, JA, Claing, A, Lavergne, J-P, Reboud, J-P, and Lefkowitz, RJ. "Beta 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2." Biochemistry 41.42 (October 22, 2002): 12850-12857.
PMID
12379128
Source
pubmed
Published In
Biochemistry
Volume
41
Issue
42
Publish Date
2002
Start Page
12850
End Page
12857

β2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the β2-adrenergic receptor (β2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the β2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the β2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with KM values approximating 1 μM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

Authors
Freeman, JLR; Gonzalo, P; Pitcher, JA; Claing, A; Lavergne, JP; Reboud, JP; Lefkowitz, RJ
MLA Citation
Freeman, JLR, Gonzalo, P, Pitcher, JA, Claing, A, Lavergne, JP, Reboud, JP, and Lefkowitz, RJ. 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2." Biochemistry 41.42 (October 22, 2002): 12850-12857.
Source
scopus
Published In
Biochemistry
Volume
41
Issue
42
Publish Date
2002
Start Page
12850
End Page
12857
DOI
10.1021/bi020145d

Seven-transmembrane receptors.

Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

Authors
Pierce, KL; Premont, RT; Lefkowitz, RJ
MLA Citation
Pierce, KL, Premont, RT, and Lefkowitz, RJ. "Seven-transmembrane receptors." Nat Rev Mol Cell Biol 3.9 (September 2002): 639-650. (Review)
PMID
12209124
Source
pubmed
Published In
Nature Reviews Molecular Cell Biology
Volume
3
Issue
9
Publish Date
2002
Start Page
639
End Page
650
DOI
10.1038/nrm908

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors.

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.

Authors
Lin, F-T; Chen, W; Shenoy, S; Cong, M; Exum, ST; Lefkowitz, RJ
MLA Citation
Lin, F-T, Chen, W, Shenoy, S, Cong, M, Exum, ST, and Lefkowitz, RJ. "Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors." Biochemistry 41.34 (August 27, 2002): 10692-10699.
PMID
12186555
Source
pubmed
Published In
Biochemistry
Volume
41
Issue
34
Publish Date
2002
Start Page
10692
End Page
10699

Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system.

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).

Authors
Zamah, AM; Delahunty, M; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Zamah, AM, Delahunty, M, Luttrell, LM, and Lefkowitz, RJ. "Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system." J Biol Chem 277.34 (August 23, 2002): 31249-31256.
PMID
12063255
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
34
Publish Date
2002
Start Page
31249
End Page
31256
DOI
10.1074/jbc.M202753200

Protein kinase A-mediated phosphorylation of the beta(2)-Adrenergic receptor regulates its coupling to G(s) and G(i) - Demonstration in a reconstituted system

Authors
Zamah, AM; Delahunty, M; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Zamah, AM, Delahunty, M, Luttrell, LM, and Lefkowitz, RJ. "Protein kinase A-mediated phosphorylation of the beta(2)-Adrenergic receptor regulates its coupling to G(s) and G(i) - Demonstration in a reconstituted system." JOURNAL OF BIOLOGICAL CHEMISTRY 277.34 (August 23, 2002): 31249-31256.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
277
Issue
34
Publish Date
2002
Start Page
31249
End Page
31256
DOI
10.1074/jbm.M202753200

Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor.

Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.

Authors
Ahn, S; Kim, J; Lucaveche, CL; Reedy, MC; Luttrell, LM; Lefkowitz, RJ; Daaka, Y
MLA Citation
Ahn, S, Kim, J, Lucaveche, CL, Reedy, MC, Luttrell, LM, Lefkowitz, RJ, and Daaka, Y. "Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor." J Biol Chem 277.29 (July 19, 2002): 26642-26651.
PMID
12011079
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
29
Publish Date
2002
Start Page
26642
End Page
26651
DOI
10.1074/jbc.M201499200

Inhibition of betaARK1 restores impaired biochemical beta-adrenergic receptor responsiveness but does not rescue CREB(A133) induced cardiomyopathy.

The myocardial beta-adrenergic receptor (betaAR) system plays a key role in dysfunctional signaling and physiology of the failing heart. Recently we described a murine model of dilated cardiomyopathy (DCM) produced by cardiac-specific expression of a dominant negative form of the CREB transcription factor (CREB(A133) mice). CREB(A133) mice display abnormalities within the betaAR signaling system including loss of inotropic reserve. Rapid desensitization of betaARs is mediated by the betaAR kinase (betaARK1), which is upregulated during heart failure. Inhibition of betaARK1 activity in the heart via expression of a peptide inhibitor (betaARKct) has been shown to enhance myocardial function and to "rescue" several animal models of heart failure. To determine the role of betaAR dysfunction in the progression of DCM in the CREB(A133) mice, we interbred them with mice expressing the betaARKct. Concurrent expression of the betaARKct peptide and CREB(A133) in mouse hearts resulted in the normalization of elevated betaARK1 levels. This biochemical change resulted in partial restoration of isoproterenol-stimulated adenylate cyclase activity as well as improvement in fractional shortening in response to betaAR stimulation. Interestingly, the progression of DCM and premature mortality was not altered. Therefore, the pathogenesis of DCM in CREB(A133) mice does not appear to involve abnormal betaAR signaling as a key element in its pathological progression and accordingly, the restoration of betaAR signaling is not sufficient to prevent the development and progression of all forms of heart failure.

Authors
Eckhart, AD; Fentzke, RC; Lepore, J; Lang, R; Lin, H; Lefkowitz, RJ; Koch, WJ; Leiden, JM
MLA Citation
Eckhart, AD, Fentzke, RC, Lepore, J, Lang, R, Lin, H, Lefkowitz, RJ, Koch, WJ, and Leiden, JM. "Inhibition of betaARK1 restores impaired biochemical beta-adrenergic receptor responsiveness but does not rescue CREB(A133) induced cardiomyopathy." J Mol Cell Cardiol 34.6 (June 2002): 669-677.
PMID
12054854
Source
pubmed
Published In
Journal of Molecular and Cellular Cardiology
Volume
34
Issue
6
Publish Date
2002
Start Page
669
End Page
677
DOI
10.1006/jmcc.2002.2007

Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ("desensitized") by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various beta-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of beta-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the beta-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from beta-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that beta-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.

Authors
Fong, AM; Premont, RT; Richardson, RM; Yu, Y-RA; Lefkowitz, RJ; Patel, DD
MLA Citation
Fong, AM, Premont, RT, Richardson, RM, Yu, Y-RA, Lefkowitz, RJ, and Patel, DD. "Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice." Proc Natl Acad Sci U S A 99.11 (May 28, 2002): 7478-7483.
Website
http://hdl.handle.net/10161/7804
PMID
12032308
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
11
Publish Date
2002
Start Page
7478
End Page
7483
DOI
10.1073/pnas.112198299

beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation.

beta-Arrestins are cytosolic proteins that mediate homologous desensitization of G protein-coupled receptors (GPCRs) by binding to agonist-occupied receptors and by uncoupling them from heterotrimeric G proteins. The recent finding that beta-arrestins bind to some mitogen-activated protein (MAP) kinases has suggested that they might also function as scaffolds for GPCR-stimulated MAP kinase activation. To define the role of beta-arrestins in the regulation of ERK MAP kinases, we examined the effect of beta-arrestin overexpression on ERK1/2 activation and nuclear signaling in COS-7 cells expressing angiotensin II type 1a receptors (AT1aRs). Expression of either beta-arrestin1 or beta-arrestin2 reduced angiotensin-stimulated phosphatidylinositol hydrolysis but paradoxically increased angiotensin-stimulated ERK1/2 phosphorylation. The increase in ERK1/2 phosphorylation in beta-arrestin-expressing cells correlated with activation of a beta-arrestin-bound pool of ERK2. The beta-arrestin-dependent increase in ERK1/2 phosphorylation was accompanied by a significant reduction in ERK1/2-mediated, Elk1-driven transcription of a luciferase reporter. Analysis of the cellular distribution of phospho-ERK1/2 by confocal immunofluorescence microscopy and cellular fractionation revealed that overexpression of beta-arrestin resulted in a significant increase in the cytosolic pool of phospho-ERK1/2 and a corresponding decrease in the nuclear pool of phospho-ERK1/2 following angiotensin stimulation. beta-Arrestin overexpression resulted in formation of a cytoplasmic pool of beta-arrestin-bound phospho-ERK, decreased nuclear translocation of phospho-ERK1/2, and inhibition of Elk1-driven luciferase transcription even when ERK1/2 was activated by overexpression of cRaf-1 in the absence of AT1aR stimulation. These data demonstrate that beta-arrestins facilitate GPCR-mediated ERK activation but inhibit ERK-dependent transcription by binding to phospho-ERK1/2, leading to its retention in the cytosol.

Authors
Tohgo, A; Pierce, KL; Choy, EW; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Tohgo, A, Pierce, KL, Choy, EW, Lefkowitz, RJ, and Luttrell, LM. "beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation." J Biol Chem 277.11 (March 15, 2002): 9429-9436.
PMID
11777902
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
11
Publish Date
2002
Start Page
9429
End Page
9436
DOI
10.1074/jbc.M106457200

Arresting developments in heptahelical receptor signaling and regulation.

It is well established that the function of most heptahelical receptors (seven-transmembrane-span receptors; 7TMRs) is tightly regulated by the desensitizing actions of arrestins. Desensitization is the waning of 7TMR-mediated signals after prolonged exposure to agonist and occurs when arrestins bind to agonist-occupied and phosphorylated receptors, uncoupling the receptors from G proteins and preventing further signaling. Recently, there has been a marked shift in the focus of research into arrestin function because it has become clear that they not only prevent signaling from 7TMRs but also initiate and direct new signals from the very 7TMRs that they desensitize.

Authors
Perry, SJ; Lefkowitz, RJ
MLA Citation
Perry, SJ, and Lefkowitz, RJ. "Arresting developments in heptahelical receptor signaling and regulation." Trends Cell Biol 12.3 (March 2002): 130-138. (Review)
PMID
11859025
Source
pubmed
Published In
Trends in Cell Biology
Volume
12
Issue
3
Publish Date
2002
Start Page
130
End Page
138

The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals.

beta-Arrestins are versatile adapter proteins that form complexes with most G-protein-coupled receptors (GPCRs) following agonist binding and phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs). They play a central role in the interrelated processes of homologous desensitization and GPCR sequestration, which lead to the termination of G protein activation. beta-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis. Recent data suggest that beta-arrestins also function as GPCR signal transducers. They can form complexes with several signaling proteins, including Src family tyrosine kinases and components of the ERK1/2 and JNK3 MAP kinase cascades. By recruiting these kinases to agonist-occupied GPCRs, beta-arrestins confer distinct signaling activities upon the receptor. beta-arrestin-Src complexes have been proposed to modulate GPCR endocytosis, to trigger ERK1/2 activation and to mediate neutrophil degranulation. By acting as scaffolds for the ERK1/2 and JNK3 cascades, beta-arrestins both facilitate GPCR-stimulated MAP kinase activation and target active MAP kinases to specific locations within the cell. Thus, their binding to GPCRs might initiate a second wave of signaling and represent a novel mechanism of GPCR signal transduction.

Authors
Luttrell, LM; Lefkowitz, RJ
MLA Citation
Luttrell, LM, and Lefkowitz, RJ. "The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals." J Cell Sci 115.Pt 3 (February 1, 2002): 455-465. (Review)
PMID
11861753
Source
pubmed
Published In
Journal of cell science
Volume
115
Issue
Pt 3
Publish Date
2002
Start Page
455
End Page
465

Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and beta-arrestin proteins.

Sequestration of G protein-coupled receptors from the cell surface is a commonly observed phenomenon following agonist-stimulation. This process is now believed to be important for receptor resensitization as well as for signal transduction. Over the years, numerous studies have aimed at understanding the molecular mechanisms underlying internalization. Proteins such as the G protein-coupled receptor kinases (GRKs) and the beta-arrestins, which were initially characterized as desensitizing molecules, have been shown to be important regulators of the endocytic process. Recently, numerous interacting partners have been identified for each of these two classes of proteins. However, the details regarding the sequence of these interactions and the cross-talk between signaling pathways containing the different protein complexes are just beginning to be uncovered. In this review, we summarize these findings and discuss the role of GRKs and beta-arrestins, two families of key regulatory proteins that regulate G protein-coupled receptor endocytosis.

Authors
Claing, A; Laporte, SA; Caron, MG; Lefkowitz, RJ
MLA Citation
Claing, A, Laporte, SA, Caron, MG, and Lefkowitz, RJ. "Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and beta-arrestin proteins." Prog Neurobiol 66.2 (February 2002): 61-79. (Review)
PMID
11900882
Source
pubmed
Published In
Progress in Neurobiology
Volume
66
Issue
2
Publish Date
2002
Start Page
61
End Page
79

G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95.

We previously reported that the beta(1)-adrenergic receptor (beta(1)AR) associates with PSD-95 through a PDZ domain-mediated interaction, by which PSD-95 modulates beta(1)AR function and facilitates the physical association of beta(1)AR with other synaptic proteins such as N-methyl-d-aspartate receptors. Here we demonstrate that beta(1)AR association with PSD-95 is regulated by G protein-coupled receptor kinase 5 (GRK5). When beta(1)AR and PSD-95 were coexpressed with either GRK2 or GRK5 in COS-7 cells, GRK5 alone dramatically decreased the association of beta(1)AR with PSD-95, although GRK2 and GRK5 both could be co-immunoprecipitated with beta(1)AR and both could enhance receptor phosphorylation in vivo. Increasing expression of GRK5 in the cells led to further decreased beta(1)AR association with PSD-95. Stimulation with the beta(1)AR agonist isoproterenol further decreased PSD-95 binding to beta(1)AR. In addition, GRK5 protein kinase activity was required for this regulatory effect since a kinase-inactive GRK5 mutant had no effect on PSD-95 binding to beta(1)AR. Moreover, the regulatory effect of GRK5 on beta(1)AR association with PSD-95 was observed only when GRK5 was expressed together with the receptor, but not when GRK5 was coexpressed with PSD-95. Thus, we propose that GRK5 regulates beta(1)AR association with PSD-95 through phosphorylation of beta(1)AR. Regulation of protein association through receptor phosphorylation may be a general mechanism used by G protein-coupled receptors that associate via PDZ domain-mediated protein/protein interactions.

Authors
Hu, LA; Chen, W; Premont, RT; Cong, M; Lefkowitz, RJ
MLA Citation
Hu, LA, Chen, W, Premont, RT, Cong, M, and Lefkowitz, RJ. "G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95." J Biol Chem 277.2 (January 11, 2002): 1607-1613.
PMID
11700307
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
2
Publish Date
2002
Start Page
1607
End Page
1613
DOI
10.1074/jbc.M107297200

Seven-transmembrane-spanning receptors and heart function.

Understanding precisely how the heart can recognize and respond to many different extracellular signalling molecules, such as neurotransmitters, hormones and growth factors, will aid the identification of new therapeutic targets through which cardiovascular diseases can be combated. In recent years, we have learned more about the complex interactions that occur between the receptors and the signalling pathways of the heart and its environment. Most of these discoveries have focused on the most common type of cardiac receptor - the seven-transmembrane-spanning receptor or G-protein-coupled receptor.

Authors
Rockman, HA; Koch, WJ; Lefkowitz, RJ
MLA Citation
Rockman, HA, Koch, WJ, and Lefkowitz, RJ. "Seven-transmembrane-spanning receptors and heart function." Nature 415.6868 (January 10, 2002): 206-212. (Review)
PMID
11805844
Source
pubmed
Published In
Nature
Volume
415
Issue
6868
Publish Date
2002
Start Page
206
End Page
212
DOI
10.1038/415206a

Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes: Differential regulation of β-arrestins 1 and 2

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo- and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either β-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only β-arrestin 2 could enhance TRHR2 internalization. The preference for β-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a β-arrestin 2 knockout or a β-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both β-arrestins. The differential β-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and β-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with β-arrestins, could also enhance TRHR2/β-arrestin I interaction. This study demonstrates that TRHR subtypes are differentially regulated by the β-arrestins and also provides the first evidence that the interactions of TRHRs with β-arrestin may be altered by hetero-oligomer formation.

Authors
Hanyaloglu, AC; Seeber, RM; Kohout, TA; Lefkowitz, RJ; Eidne, KA
MLA Citation
Hanyaloglu, AC, Seeber, RM, Kohout, TA, Lefkowitz, RJ, and Eidne, KA. "Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes: Differential regulation of β-arrestins 1 and 2." Journal of Biological Chemistry 277.52 (2002): 50422-50430.
PMID
12393857
Source
scival
Published In
Journal of Biological Chemistry
Volume
277
Issue
52
Publish Date
2002
Start Page
50422
End Page
50430
DOI
10.1074/jbc.M209340200

Regulation of G protein-coupled receptor signaling by scaffold proteins

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

Authors
Hall, RA; Lefkowitz, RJ
MLA Citation
Hall, RA, and Lefkowitz, RJ. "Regulation of G protein-coupled receptor signaling by scaffold proteins." Circulation Research 91.8 (2002): 672-680.
Website
http://hdl.handle.net/10161/5917
PMID
12386143
Source
scival
Published In
Circulation Research
Volume
91
Issue
8
Publish Date
2002
Start Page
672
End Page
680
DOI
10.1161/01.RES.0000037000.74258.03

β2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the β2-adrenergic receptor (β2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the β2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the β2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with KM values approximating 1 μM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

Authors
Freeman, JLR; Gonzalo, P; Pitcher, JA; Claing, A; Lavergne, J-P; Reboud, J-P; Lefkowitz, RJ
MLA Citation
Freeman, JLR, Gonzalo, P, Pitcher, JA, Claing, A, Lavergne, J-P, Reboud, J-P, and Lefkowitz, RJ. 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2." Biochemistry 41.42 (2002): 12850-12857.
Source
scival
Published In
Biochemistry
Volume
41
Issue
42
Publish Date
2002
Start Page
12850
End Page
12857
DOI
10.1021/bi020145d

β-arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation

The widely expressed β-arrestin isoforms 1 and 2 bind phosphorylated G protein-coupled receptors (GPCRs) and mediate desensitization and internalization. Phosphorylation of protease-activated receptor-1 (PAR1 ), a GPCR for thrombin, is important for desensitization and internalization, however, the role of β-arrestins in signaling and trafficking of PAR1 remains unknown. To assess β-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from β-arrestin (βarr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both βarr1 and βarr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only βarr1 PAR1 desensitization was also significantly impaired compared with βarr2-lacking or wild-type cells. In wild-type MEFs, activated PAR1 was internalized through a dynamin- and clathrin-dependent pathway and degraded. Surprisingly, in cells lacking both βarr1 and βarr2 activated PAR1 was similarly internalized through a dynamin- and clathrin-dependent pathway and degraded, whereas the β2-adrenergic receptor (β2-AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and β-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but β-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual β-arrestin isoforms can differentially regulate GPCR desensitization and further reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.

Authors
Paing, MM; Stutts, AB; Kohout, TA; Lefkowitz, RJ; Trejo, J
MLA Citation
Paing, MM, Stutts, AB, Kohout, TA, Lefkowitz, RJ, and Trejo, J. "β-arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation." Journal of Biological Chemistry 277.2 (2002): 1292-1300.
PMID
11694535
Source
scival
Published In
The Journal of biological chemistry
Volume
277
Issue
2
Publish Date
2002
Start Page
1292
End Page
1300
DOI
10.1074/jbc.M109160200

The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals

β-arrestins are versatile adapter proteins that form complexes with most G-protein-coupled receptors (GPCRs) following agonist binding and phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs). They play a central role in the interrelated processes of homologous desensitization and GPCR sequestration, which lead to the termination of G protein activation. β-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrincoated pits for endocytosis. Recent data suggest that β-arrestins also function as GPCR signal transducers. They can form complexes with several signaling proteins, including Src family tyrosine kinases and components of the ERK1/2 and JNK3 MAP kinase cascades. By recruiting these kinases to agonist-occupied GPCRs, β-arrestins confer distinct signaling activities upon the receptor. β-arrestin-Src complexes have been proposed to modulate GPCR endocytosis, to trigger ERK1/2 activation and to mediate neutrophil degranulation. By acting as scaffolds for the ERK1/2 and JNK3 cascades, β-arrestins both facilitate GPCR-stimulated MAP kinase activation and target active MAP kinases to specific locations within the cell. Thus, their binding to GPCRs might initiate a second wave of signaling and represent a novel mechanism of GPCR signal transduction.

Authors
Luttrell, LM; Lefkowitz, RJ
MLA Citation
Luttrell, LM, and Lefkowitz, RJ. "The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals." Journal of Cell Science 115.3 (2002): 455-465.
Website
http://hdl.handle.net/10161/7805
Source
scival
Published In
Journal of Cell Science
Volume
115
Issue
3
Publish Date
2002
Start Page
455
End Page
465

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Authors
Chen, W; Hu, LA; Semenov, MV; Yanagawa, S; Kikuchi, A; Lefkowitz, RJ; Miller, WE
MLA Citation
Chen, W, Hu, LA, Semenov, MV, Yanagawa, S, Kikuchi, A, Lefkowitz, RJ, and Miller, WE. "beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins." Proc Natl Acad Sci U S A 98.26 (December 18, 2001): 14889-14894.
Website
http://hdl.handle.net/10161/7809
PMID
11742073
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
26
Publish Date
2001
Start Page
14889
End Page
14894
DOI
10.1073/pnas.211572798

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.

Authors
Cong, M; Perry, SJ; Hu, LA; Hanson, PI; Claing, A; Lefkowitz, RJ
MLA Citation
Cong, M, Perry, SJ, Hu, LA, Hanson, PI, Claing, A, and Lefkowitz, RJ. "Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling." J Biol Chem 276.48 (November 30, 2001): 45145-45152.
PMID
11577089
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
48
Publish Date
2001
Start Page
45145
End Page
45152
DOI
10.1074/jbc.M106087200

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.

Authors
Kohout, TA; Takaoka, H; McDonald, PH; Perry, SJ; Mao, L; Lefkowitz, RJ; Rockman, HA
MLA Citation
Kohout, TA, Takaoka, H, McDonald, PH, Perry, SJ, Mao, L, Lefkowitz, RJ, and Rockman, HA. "Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice." Circulation 104.20 (November 13, 2001): 2485-2491.
Website
http://hdl.handle.net/10161/5906
PMID
11705829
Source
pubmed
Published In
Circulation
Volume
104
Issue
20
Publish Date
2001
Start Page
2485
End Page
2491

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin.

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.

Authors
Shenoy, SK; McDonald, PH; Kohout, TA; Lefkowitz, RJ
MLA Citation
Shenoy, SK, McDonald, PH, Kohout, TA, and Lefkowitz, RJ. "Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin." Science 294.5545 (November 9, 2001): 1307-1313.
PMID
11588219
Source
pubmed
Published In
Science
Volume
294
Issue
5545
Publish Date
2001
Start Page
1307
End Page
1313
DOI
10.1126/science.1063866

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis.

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

Authors
Claing, A; Chen, W; Miller, WE; Vitale, N; Moss, J; Premont, RT; Lefkowitz, RJ
MLA Citation
Claing, A, Chen, W, Miller, WE, Vitale, N, Moss, J, Premont, RT, and Lefkowitz, RJ. "beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis." J Biol Chem 276.45 (November 9, 2001): 42509-42513.
PMID
11533043
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
45
Publish Date
2001
Start Page
42509
End Page
42513
DOI
10.1074/jbc.M108399200

Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors.

In the classical model of G-protein-coupled receptor (GPCR) regulation, arrestins terminate receptor signalling. After receptor activation, arrestins desensitize phosphorylated GPCRs, blocking further activation and initiating receptor internalization. This function of arrestins is exemplified by studies on the role of arrestins in the development of tolerance to, but not dependence on, morphine. Arrestins also link GPCRs to several signalling pathways, including activation of the non-receptor tyrosine kinase SRC and mitogen-activated protein kinase. In these cascades, arrestins function as adaptors and scaffolds, bringing sequentially acting kinases into proximity with each other and the receptor. The signalling roles of arrestins have been expanded even further with the discovery that the formation of stable receptor-arrestin complexes initiates photoreceptor apoptosis in Drosophila, leading to retinal degeneration. Here we review our current understanding of arrestin function, discussing both its classical and newly discovered roles.

Authors
Pierce, KL; Lefkowitz, RJ
MLA Citation
Pierce, KL, and Lefkowitz, RJ. "Classical and new roles of beta-arrestins in the regulation of G-protein-coupled receptors." Nat Rev Neurosci 2.10 (October 2001): 727-733. (Review)
PMID
11584310
Source
pubmed
Published In
Nature Reviews Neuroscience
Volume
2
Issue
10
Publish Date
2001
Start Page
727
End Page
733
DOI
10.1038/35094577

2001: An AAP (Association of American Physicians) odyssey.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "2001: An AAP (Association of American Physicians) odyssey." J Clin Invest 108.7 (October 2001): s9-s13.
PMID
11669075
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
108
Issue
7
Publish Date
2001
Start Page
s9
End Page
s13

Beta-Arrestins: new roles in regulating heptahelical receptors' functions.

The last few years have seen a marked expansion in appreciation of the diversity of roles played by the betaArrestins in regulating GPCR functions. Originally discovered as molecules that desensitize such receptors, the roles of betaArrestins have expanded to include acting as signalling adapters or intermediates that recruit other key molecules to the GPCRs in an agonist-regulated fashion. For example, interactions with components of the endocytic machinery, such as clathrin, the adapter protein AP-2 and the N-ethylmaleimide sensitive fusion protein (NSF), demonstrate the ability of betaArrestins to act as adapters to facilitate the clathrin-mediated endocytosis of certain members of the GPCR family. BetaArrestins have also been shown to serve as signalling molecules. The Ras-dependent activation of ERK1/2 may involve the betaArrestin-dependent recruitment of c-Src to the beta2-adrenergic receptor (beta2-AR). More recently, betaArrestins have been shown to act as molecular scaffolds that coordinate the assembly of certain MAP kinase complexes that lead to the stimulation of either ERK1/2 or JNK3. Finally, long-term accumulation of arrestin-rhodopsin complexes, in photoreceptor cells has been shown to trigger apoptosis.

Authors
McDonald, PH; Lefkowitz, RJ
MLA Citation
McDonald, PH, and Lefkowitz, RJ. "Beta-Arrestins: new roles in regulating heptahelical receptors' functions." Cell Signal 13.10 (October 2001): 683-689. (Review)
PMID
11602178
Source
pubmed
Published In
Cellular Signalling
Volume
13
Issue
10
Publish Date
2001
Start Page
683
End Page
689

Altered bone mass and microarchitecture in beta-arrestin2 KO mice.

Authors
Ferrari, SL; Lin, F; Lefkowitz, RJ; Bouxsein, ML
MLA Citation
Ferrari, SL, Lin, F, Lefkowitz, RJ, and Bouxsein, ML. "Altered bone mass and microarchitecture in beta-arrestin2 KO mice." JOURNAL OF BONE AND MINERAL RESEARCH 16 (September 2001): S158-S158.
Source
wos-lite
Published In
Journal of Bone and Mineral Research
Volume
16
Publish Date
2001
Start Page
S158
End Page
S158

Regulation of myocardial betaARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing alpha1B-adrenergic receptors.

OBJECTIVES: Using a transgenic mouse model of myocardial-targeted overexpression of the wild-type alpha1B adrenergic receptor (AR) (Tg alpha43), we studied the role of the betaAR kinase (betaARK1) in the evolution of myocardial hypertrophy and its transition to heart failure (HF). BACKGROUND: Increased myocardial expression of betaARK1 has been shown to be associated with HF and certain models of hypertrophy. METHODS: Tg alpha43 mice and their nontransgenic littermate controls were treated with the alpha1AR agonist phenylephrine (PE) for 3, 7 or 14 days to characterize the cardiac consequences. RESULTS: Nontransgenic littermate control mice treated for 14 days with PE display cardiac hypertrophy with no increase in betaARK1 expression. However, Tg alpha43 animals show a reduced tolerance to 14-day PE treatment, demonstrated by reduced survival and severe cardiac hypertrophy. Moreover, PE treatment for three and seven days in Tg alpha43 mice resulted in an exaggerated hypertrophic response accompanied by significant cardiac biochemical abnormalities that are normally associated with HF, including fetal gene expression, reduced betaAR density and enhanced betaARK1 expression. We also found reduced myocardial stores of the sympathetic neurotransmitter neuropeptide Y. CONCLUSIONS: These data suggest that PE-treated Tg alpha43 mice have chronic activation of the cardiac sympathetic nervous system, which may be responsible for the appearance of apparent maladaptive hypertrophy with an evolution towards HF and sudden death. Thus, the cardiac phenotypes found in these mice are not the direct result of enhanced alpha1B AR signaling and suggest that betaARK1 is a key molecule in the transition of myocardial hypertrophy to HF.

Authors
Iaccarino, G; Keys, JR; Rapacciuolo, A; Shotwell, KF; Lefkowitz, RJ; Rockman, HA; Koch, WJ
MLA Citation
Iaccarino, G, Keys, JR, Rapacciuolo, A, Shotwell, KF, Lefkowitz, RJ, Rockman, HA, and Koch, WJ. "Regulation of myocardial betaARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing alpha1B-adrenergic receptors." J Am Coll Cardiol 38.2 (August 2001): 534-540.
PMID
11499749
Source
pubmed
Published In
JACC - Journal of the American College of Cardiology
Volume
38
Issue
2
Publish Date
2001
Start Page
534
End Page
540

Identification of a motif in the carboxyl terminus of beta -arrestin2 responsible for activation of JNK3.

Accumulating evidence indicates that the beta-arrestins act as scaffold molecules that couple G-protein-coupled receptors to mitogen-activated protein (MAP) kinase signaling pathways. Recently, we identified the c-Jun N-terminal kinase 3 (JNK3) as a beta-arrestin2-interacting protein in yeast-two hybrid and co-immunoprecipitation studies. Beta-arrestin2 acts as a scaffold to enhance signaling to JNK3 stimulated by overexpression of the MAP3 kinase ASK1 or by agonist activation of the angiotensin 1A receptor. Whereas beta-arrestin2 is a very strong activator of JNK3 signaling, beta-arrestin1 is very weak in this regard. The data also indicate that the specific step enhanced by beta-arrestin2 involves phosphorylation of JNK3 by the MAP2 kinase MKK4. We reasoned that defining the region (or domain) in beta-arrestin2 responsible for high level JNK3 activation would provide insight into the mechanism by which beta-arrestin2 enhances the activity of this signaling pathway. Using chimeric beta-arrestins, we have determined that sequences in the carboxyl-terminal region of beta-arrestin2 are important for the enhancement of JNK3 phosphorylation. More detailed analysis of the carboxyl-terminal domains of the beta-arrestins indicated that beta-arrestin2, but not beta-arrestin1, contains a sequence (RRSLHL) highly homologous to the conserved docking motif present in many MAP kinase-binding proteins. Replacement of the beta-arrestin2 RRS residues with the corresponding KP residues present in beta-arrestin1 dramatically reduced both JNK3 interaction and enhancement of JNK3 phosphorylation. Conversely, replacement of the KP residues in beta-arrestin1 with RRS significantly increased both JNK3 binding and enhancement of JNK3 phosphorylation. These results delineate a mechanism by which beta-arrestin2 functions as a scaffold protein in the JNK3 signaling pathway and implicate the conserved docking site in beta-arrestin2 as an important factor in binding JNK3 and stimulating the phosphorylation of JNK3 by MKK4.

Authors
Miller, WE; McDonald, PH; Cai, SF; Field, ME; Davis, RJ; Lefkowitz, RJ
MLA Citation
Miller, WE, McDonald, PH, Cai, SF, Field, ME, Davis, RJ, and Lefkowitz, RJ. "Identification of a motif in the carboxyl terminus of beta -arrestin2 responsible for activation of JNK3." J Biol Chem 276.30 (July 27, 2001): 27770-27777.
PMID
11356842
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
30
Publish Date
2001
Start Page
27770
End Page
27777
DOI
10.1074/jbc.M102264200

Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding.

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.

Authors
Pierce, KL; Tohgo, A; Ahn, S; Field, ME; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Pierce, KL, Tohgo, A, Ahn, S, Field, ME, Luttrell, LM, and Lefkowitz, RJ. "Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding." J Biol Chem 276.25 (June 22, 2001): 23155-23160.
PMID
11290747
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
25
Publish Date
2001
Start Page
23155
End Page
23160
DOI
10.1074/jbc.M101303200

Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure.

Chronic human heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased levels of betaAR kinase 1 (betaARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of betaARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of betaARK1 (betaARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca(2+)-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 +/- 1 weeks). In contrast, CSQ/betaARKct mice exhibited a significant increase in mean survival age (15 +/- 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/betaARKct, left ventricular end diastolic dimension 5.60 +/- 0.17 mm vs. 4.19 +/- 0.09 mm, P < 0.005; % fractional shortening, 15 +/- 2 vs. 36 +/- 2, P < 0.005). The enhancement of the survival rate in CSQ/betaARKct mice was substantially potentiated by chronic treatment with the betaAR antagonist metoprolol (CSQ/betaARKct nontreated vs. CSQ/betaARKct metoprolol treated, 15 +/- 1 weeks vs. 25 +/- 2 weeks, P < 0.0001). Thus, overexpression of the betaARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with beta-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of betaARK1 inhibition.

Authors
Harding, VB; Jones, LR; Lefkowitz, RJ; Koch, WJ; Rockman, HA
MLA Citation
Harding, VB, Jones, LR, Lefkowitz, RJ, Koch, WJ, and Rockman, HA. "Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure." Proc Natl Acad Sci U S A 98.10 (May 8, 2001): 5809-5814.
Website
http://hdl.handle.net/10161/7806
PMID
11331748
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
10
Publish Date
2001
Start Page
5809
End Page
5814
DOI
10.1073/pnas.091102398

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

Authors
Cong, M; Perry, SJ; Lin, FT; Fraser, ID; Hu, LA; Chen, W; Pitcher, JA; Scott, JD; Lefkowitz, RJ
MLA Citation
Cong, M, Perry, SJ, Lin, FT, Fraser, ID, Hu, LA, Chen, W, Pitcher, JA, Scott, JD, and Lefkowitz, RJ. "Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79." J Biol Chem 276.18 (May 4, 2001): 15192-15199.
PMID
11278469
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
18
Publish Date
2001
Start Page
15192
End Page
15199
DOI
10.1074/jbc.M009130200

Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking.

beta-arrestins play previously unsuspected and important roles as adapters and scaffolds that localize signaling proteins to ligand-activated G-protein-coupled receptors. As with the paradigmatic role of the beta-arrestins in uncoupling receptors from G proteins (desensitization), these novel functions involve the interaction of beta-arrestin with phosphorylated heptahelical receptors. beta-arrestins interact with at least two main classes of signaling proteins. First, interaction with molecules such as clathrin, AP-2 and NSF directs the clathrin-mediated internalization of G-protein-coupled receptors. Second, interaction with molecules such as Src, Raf, Erk, ASK1 and JNK3 appears to regulate several pathways that result in the activation of MAP kinases. These recent discoveries indicate that the beta-arrestins play widespread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from heptahelical receptors. It is likely that additional roles for the beta-arrestins remain to be discovered.

Authors
Miller, WE; Lefkowitz, RJ
MLA Citation
Miller, WE, and Lefkowitz, RJ. "Expanding roles for beta-arrestins as scaffolds and adapters in GPCR signaling and trafficking." Curr Opin Cell Biol 13.2 (April 2001): 139-145. (Review)
PMID
11248546
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
13
Issue
2
Publish Date
2001
Start Page
139
End Page
145

New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades.

Activation of classical second messenger cascades cannot fully explain the recently appreciated roles of heptahelical, or G-protein coupled receptors (GPCRs), in stimulation of mitogen activated protein kinase (MAPK) cascades. Rather, several distinct signaling mechanisms appear to contribute to GPCR-mediated MAPK activation. These include transactivation of the Epidermal Growth Factor Receptor (EGFR) via the autocrine/paracrine release of EGF-like ligands at the cell surface and scaffolding of MAPK cascades. A significant advance in the understanding of how GPCRs activate MAPK cascades is the discovery that beta-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade. Together, these novel mechanisms of GPCR-mediated MAPK regulation may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs and thereby determine the consequences of GPCR stimulation with respect to transcriptional activation, cell proliferation and apoptosis.

Authors
Pierce, KL; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Pierce, KL, Luttrell, LM, and Lefkowitz, RJ. "New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades." Oncogene 20.13 (March 26, 2001): 1532-1539. (Review)
PMID
11313899
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
20
Issue
13
Publish Date
2001
Start Page
1532
End Page
1539
DOI
10.1038/sj.onc.1204184

In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.

BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.

Authors
Shah, AS; White, DC; Emani, S; Kypson, AP; Lilly, RE; Wilson, K; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Shah, AS, White, DC, Emani, S, Kypson, AP, Lilly, RE, Wilson, K, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction." Circulation 103.9 (March 6, 2001): 1311-1316.
Website
http://hdl.handle.net/10161/5905
PMID
11238278
Source
pubmed
Published In
Circulation
Volume
103
Issue
9
Publish Date
2001
Start Page
1311
End Page
1316

Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

Authors
Luttrell, LM; Roudabush, FL; Choy, EW; Miller, WE; Field, ME; Pierce, KL; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Roudabush, FL, Choy, EW, Miller, WE, Field, ME, Pierce, KL, and Lefkowitz, RJ. "Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds." Proc Natl Acad Sci U S A 98.5 (February 27, 2001): 2449-2454.
Website
http://hdl.handle.net/10161/7807
PMID
11226259
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
5
Publish Date
2001
Start Page
2449
End Page
2454
DOI
10.1073/pnas.041604898

Arrestins as signaling molecules involved in apoptotic pathways: a real eye opener.

Recent data suggest that internalized receptor and arrestin complexes are actively involved in signal transduction. Miller and Lefkowitz discuss evidence from the Drosophila visual system that suggests that intracellular rhodopsin and arrestin2 complexes induce apoptosis. Experiments with activated mammalian G protein-coupled receptor and arrestin complexes point to a mechanism by which proliferative or proapoptotic signals can be mediated largely independent from G protein activation.

Authors
Miller, WE; Lefkowitz, RJ
MLA Citation
Miller, WE, and Lefkowitz, RJ. "Arrestins as signaling molecules involved in apoptotic pathways: a real eye opener. (Published online)" Sci STKE 2001.69 (February 13, 2001): pe1-. (Review)
PMID
11752639
Source
pubmed
Published In
Sciences STKE [electronic resource] : signal transduction knowledge environment
Volume
2001
Issue
69
Publish Date
2001
Start Page
pe1
DOI
10.1126/stke.2001.69.pe1

beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking.

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Authors
Kohout, TA; Lin, FS; Perry, SJ; Conner, DA; Lefkowitz, RJ
MLA Citation
Kohout, TA, Lin, FS, Perry, SJ, Conner, DA, and Lefkowitz, RJ. "beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking." Proc Natl Acad Sci U S A 98.4 (February 13, 2001): 1601-1606.
Website
http://hdl.handle.net/10161/7808
PMID
11171997
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
4
Publish Date
2001
Start Page
1601
End Page
1606
DOI
10.1073/pnas.041608198

β-Arrestin-mediated Recruitment of the Src Family Kinase Yes Mediates Endothelin-1-stimulated Glucose Transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein αq/11 (Gαq/11), leading to Gαq/11 tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Gαq/11 tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Gαq/11 tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that β-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that β-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of β-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Gαq/11 in response to ET-1 stimulation, and 2) β-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Gα q/11 with subsequent glucose transport stimulation.

Authors
Imamura, T; Huang, J; Dalle, S; Ugi, S; Usui, I; Luttrell, LM; Miller, WE; Lefkowitz, RJ; Olefsky, JM
MLA Citation
Imamura, T, Huang, J, Dalle, S, Ugi, S, Usui, I, Luttrell, LM, Miller, WE, Lefkowitz, RJ, and Olefsky, JM. "β-Arrestin-mediated Recruitment of the Src Family Kinase Yes Mediates Endothelin-1-stimulated Glucose Transport." Journal of Biological Chemistry 276.47 (2001): 43663-43667.
PMID
11546805
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
47
Publish Date
2001
Start Page
43663
End Page
43667
DOI
10.1074/jbc.M105364200

Incidence of congenital heart disease [2]

Authors
Sissman, NJ; Willerson, JT; Lefkowitz, RJ
MLA Citation
Sissman, NJ, Willerson, JT, and Lefkowitz, RJ. "Incidence of congenital heart disease [2]." Journal of the American Medical Association 285.20 (2001): 2579-2580.
PMID
11368725
Source
scival
Published In
Journal of the American Medical Association
Volume
285
Issue
20
Publish Date
2001
Start Page
2579
End Page
2580

Prospects for cardiovascular research

Cardiovascular disease is the greatest threat to human life and health. The past decade has seen remarkable progress in clinical and basic cardiovascular research, and many areas of opportunity are promising. The pace of current progress in clinical and basic research is such that remarkable improvement in the quality and length of life for those at risk for cardiovascular disease is likely.

Authors
Lefkowitz, RJ; Willerson, JT
MLA Citation
Lefkowitz, RJ, and Willerson, JT. "Prospects for cardiovascular research." Journal of the American Medical Association 285.5 (2001): 581-587.
PMID
11176863
Source
scival
Published In
JAMA : the journal of the American Medical Association
Volume
285
Issue
5
Publish Date
2001
Start Page
581
End Page
587

Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes

The goal of this study was to determine whether β1-adrenergic receptor (AR) and β2-AR differ in regulating cardiomyocyte survival and apoptosis and if so, to explore underlying mechanisms. One potential mechanism is that cardiac β2-AR can activate both Gs and Gi proteins, whereas cardiac β1-AR couples only to Gs. To avoid complicated crosstalk between β-AR subtypes, we expressed β1-AR or β2-AR individually in adult β1/β2-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of β1-AR, but not β2-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, β2-AR (but not β1-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting Gi, Gβγ, or phosphoinositide 3 kinase (Pl3K) with pertussis toxin, βARK-ct (a peptide inhibitor of Gβγ), or LY294002, respectively. This indicates that β2-AR activates Akt via a Gi-Gβγ-Pl3K pathway. More importantly, inhibition of the Gi-Gβγ-Pl3K-Akt pathway converts β2-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, β2-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the Gi-Gβγ-Pl3K-Akt signaling pathway.

Authors
Zhu, W-Z; Zheng, M; Koch, WJ; Lefkowitz, RJ; Kobilka, BK; Xiao, R-P
MLA Citation
Zhu, W-Z, Zheng, M, Koch, WJ, Lefkowitz, RJ, Kobilka, BK, and Xiao, R-P. "Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes." Proceedings of the National Academy of Sciences of the United States of America 98.4 (2001): 1607-1612.
Website
http://hdl.handle.net/10161/7810
PMID
11171998
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
4
Publish Date
2001
Start Page
1607
End Page
1612
DOI
10.1073/pnas.98.4.1607

Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including β-adrenergic receptor (βAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in βAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the β2AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression ora peptide inhibitor of the βAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of βAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Authors
Freeman, K; Lerman, I; Kranias, EG; Bohlmeyer, T; Bristow, MR; Lefkowitz, RJ; Iaccarino, G; Koch, WJ; Leinwand, LA
MLA Citation
Freeman, K, Lerman, I, Kranias, EG, Bohlmeyer, T, Bristow, MR, Lefkowitz, RJ, Iaccarino, G, Koch, WJ, and Leinwand, LA. "Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy." Journal of Clinical Investigation 107.8 (2001): 967-974.
Website
http://hdl.handle.net/10161/5924
PMID
11306600
Source
scival
Published In
Journal of Clinical Investigation
Volume
107
Issue
8
Publish Date
2001
Start Page
967
End Page
974
DOI
10.1172/JCI12083

βArrestins - New roles in regulating heptahelical receptors' functions

The last few years have seen a marked expansion in appreciation of the diversity of roles played by the βArrestins in regulating GPCR functions. Originally discovered as molecules that desensitize such receptors, the roles of βArrestins have expanded to include acting as signalling adapters or intermediates that recruit other key molecules to the GPCRs in an agonist-regulated fashion. For example, interactions with components of the endocytic machinery, such as clathrin, the adapter protein AP-2 and the N-ethylmaleimide sensitive fusion protein (NSF), demonstrate the ability of βArrestins to act as adapters to facilitate the clathrin-mediated endocytosis of certain members of the GPCR family. βArrestins have also been shown to serve as signalling molecules. The Ras-dependent activation of ERK1/2 may involve the βArrestin-dependent recruitment of c-Src to the β2-adrenergic receptor (β2-AR). More recently, βArrestins have been shown to act as molecular scaffolds that coordinate the assembly of certain MAP kinase complexes that lead to the stimulation of either ERK1/2 or JNK3. Finally, long-term accumulation of arrestin-rhodopsin complexes, in photoreceptor cells has been shown to trigger apoptosis. © 2001 Elsevier Science Inc. All rights reserved.

Authors
McDonald, PH; Lefkowitz, RJ
MLA Citation
McDonald, PH, and Lefkowitz, RJ. "βArrestins - New roles in regulating heptahelical receptors' functions." Cellular Signalling 13.10 (2001): 683-689.
Source
scival
Published In
Cellular Signalling
Volume
13
Issue
10
Publish Date
2001
Start Page
683
End Page
689
DOI
10.1016/S0898-6568(01)00203-0

Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation.

Both beta(2)- and beta(3)-adrenergic receptors (ARs) are able to activate the extracellular signal-regulated kinase (ERK) pathway. We previously showed that c-Src is required for ERK activation by beta(2)AR and that it is recruited to activated beta(2)AR through binding of the Src homology 3 (SH3) domain to proline-rich regions of the adapter protein beta-arrestin1. Despite the absence of sites for phosphorylation and beta-arrestin binding, ERK activation by beta(3)AR still requires c-Src. Agonist activation of beta(2)AR, but not beta(3)AR, led to redistribution of green fluorescent protein-tagged beta-arrestin to the plasma membrane. In beta-arrestin-deficient COS-7 cells, beta-agonist-dependent co-precipitation of c-Src with the beta(2)AR required exogenous beta-arrestin, but activated beta(3)AR co-precipitated c-Src in the absence or presence of beta-arrestin. ERK activation and Src co-precipitation with beta(3)AR also occurred in adipocytes in an agonist-dependent and pertussis toxin-sensitive manner. Protein interaction studies show that the beta(3)AR interacts directly with the SH3 domain of Src through proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus. ERK activation and Src co-precipitation were abolished in cells expressing point mutations in these PXXP motifs. Together, these data describe a novel mechanism of ERK activation by a G protein-coupled receptor in which the intracellular domains directly recruit c-Src.

Authors
Cao, W; Luttrell, LM; Medvedev, AV; Pierce, KL; Daniel, KW; Dixon, TM; Lefkowitz, RJ; Collins, S
MLA Citation
Cao, W, Luttrell, LM, Medvedev, AV, Pierce, KL, Daniel, KW, Dixon, TM, Lefkowitz, RJ, and Collins, S. "Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation." J Biol Chem 275.49 (December 8, 2000): 38131-38134.
PMID
11013230
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
49
Publish Date
2000
Start Page
38131
End Page
38134
DOI
10.1074/jbc.C000592200

beta 1-adrenergic receptor association with PSD-95. Inhibition of receptor internalization and facilitation of beta 1-adrenergic receptor interaction with N-methyl-D-aspartate receptors.

The beta(1)-adrenergic receptor (beta(1)AR) is the most abundant subtype of beta-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal beta(1)AR-interacting proteins, we screened a rat brain cDNA library using the beta(1)AR carboxyl terminus (beta(1)AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the beta(1)AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the beta(1)AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length beta(1)AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between beta(1)AR and PSD-95 is mediated by the last few amino acids of the beta(1)AR, and mutation of the beta(1)AR carboxyl terminus eliminated the binding and disrupted the co-localization of the beta(1)AR and PSD-95 in cells. Agonist-induced internalization of the beta(1)AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the beta(1)AR or beta(1)AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the beta(1)AR and N-methyl-d-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific beta(1)AR binding partner that modulates beta(1)AR function and facilitates physical association of the beta(1)AR with synaptic proteins, such as the N-methyl-d-aspartate receptors, which are known to be regulated by beta(1)AR stimulation.

Authors
Hu, LA; Tang, Y; Miller, WE; Cong, M; Lau, AG; Lefkowitz, RJ; Hall, RA
MLA Citation
Hu, LA, Tang, Y, Miller, WE, Cong, M, Lau, AG, Lefkowitz, RJ, and Hall, RA. "beta 1-adrenergic receptor association with PSD-95. Inhibition of receptor internalization and facilitation of beta 1-adrenergic receptor interaction with N-methyl-D-aspartate receptors." J Biol Chem 275.49 (December 8, 2000): 38659-38666.
PMID
10995758
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
49
Publish Date
2000
Start Page
38659
End Page
38666
DOI
10.1074/jbc.M005938200

β1-adrenergic receptor association with PSD-95: Inhibition of receptor internalization and facilitation of β1-adrenergic receptor interaction with N-methyl-D-aspartate receptors

The β1-adrenergic receptor (β1AR) is the most abundant subtype of β-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal β1AR-interacting proteins, we screened a rat brain cDNA library using the β1AR carboxyl terminus (β1AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the β1AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the β1AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length β1AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between β1AR and PSD-95 is mediated by the last few amino acids of the β1AR, and mutation of the β1AR carboxyl terminus eliminated the binding and disrupted the co-localization of the β1AR and PSD-95 in cells. Agonist-induced internalization of the β1AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the β1AR or β1AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the β1AR and N-methyl-D-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific β1AR binding partner that modulates β1AR function and facilitates physical association of the β1AR with synaptic proteins, such as the N-methyl-D-aspartate receptors, which are known to be regulated by β1AR stimulation.

Authors
Hu, LA; Tang, Y; Miller, WE; Cong, M; Lau, AG; Lefkowitz, RJ; Hall, RA
MLA Citation
Hu, LA, Tang, Y, Miller, WE, Cong, M, Lau, AG, Lefkowitz, RJ, and Hall, RA. 1-adrenergic receptor association with PSD-95: Inhibition of receptor internalization and facilitation of β1-adrenergic receptor interaction with N-methyl-D-aspartate receptors." Journal of Biological Chemistry 275.49 (December 8, 2000): 38659-38666.
Source
scopus
Published In
The Journal of biological chemistry
Volume
275
Issue
49
Publish Date
2000
Start Page
38659
End Page
38666
DOI
10.1074/jbc.M005938200

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.

Authors
Bohn, LM; Gainetdinov, RR; Lin, FT; Lefkowitz, RJ; Caron, MG
MLA Citation
Bohn, LM, Gainetdinov, RR, Lin, FT, Lefkowitz, RJ, and Caron, MG. "Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence." Nature 408.6813 (December 7, 2000): 720-723.
PMID
11130073
Source
pubmed
Published In
Nature
Volume
408
Issue
6813
Publish Date
2000
Start Page
720
End Page
723
DOI
10.1038/35047086

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Authors
McDonald, PH; Chow, CW; Miller, WE; Laporte, SA; Field, ME; Lin, FT; Davis, RJ; Lefkowitz, RJ
MLA Citation
McDonald, PH, Chow, CW, Miller, WE, Laporte, SA, Field, ME, Lin, FT, Davis, RJ, and Lefkowitz, RJ. "Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3." Science 290.5496 (November 24, 2000): 1574-1577.
PMID
11090355
Source
pubmed
Published In
Science
Volume
290
Issue
5496
Publish Date
2000
Start Page
1574
End Page
1577

Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.

Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.

Authors
Maudsley, S; Zamah, AM; Rahman, N; Blitzer, JT; Luttrell, LM; Lefkowitz, RJ; Hall, RA
MLA Citation
Maudsley, S, Zamah, AM, Rahman, N, Blitzer, JT, Luttrell, LM, Lefkowitz, RJ, and Hall, RA. "Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity." Mol Cell Biol 20.22 (November 2000): 8352-8363.
Website
http://hdl.handle.net/10161/7813
PMID
11046132
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
22
Publish Date
2000
Start Page
8352
End Page
8363

A novel scaffolding role of beta arrestin2 in angiotensin II AT1 receptor activation of JNK3

Authors
Field, ME; McDonald, PH; Miller, WE; Lefkowitz, RJ
MLA Citation
Field, ME, McDonald, PH, Miller, WE, and Lefkowitz, RJ. "A novel scaffolding role of beta arrestin2 in angiotensin II AT1 receptor activation of JNK3." CIRCULATION 102.18 (October 31, 2000): 222-222.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
222
End Page
222

Enhanced cardiac contractility in transgenic mice overexpressing the human beta 3 adrenergic receptor

Authors
Kohout, TA; Takaoka, H; McDonald, PH; Perry, SJ; Mao, L; Lefkowitz, RJ; Rockman, HA
MLA Citation
Kohout, TA, Takaoka, H, McDonald, PH, Perry, SJ, Mao, L, Lefkowitz, RJ, and Rockman, HA. "Enhanced cardiac contractility in transgenic mice overexpressing the human beta 3 adrenergic receptor." CIRCULATION 102.18 (October 31, 2000): 276-276.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
276
End Page
276

Role of nitric oxide synthase (NOS) in protection from myocardial ischemic injury in female, vs. male, beta(2)-adrenergic receptor (beta(2)AR) overexpressor mice

Authors
Cross, HR; Murphy, E; Lefkowitz, RJ; Steenbergen, C
MLA Citation
Cross, HR, Murphy, E, Lefkowitz, RJ, and Steenbergen, C. "Role of nitric oxide synthase (NOS) in protection from myocardial ischemic injury in female, vs. male, beta(2)-adrenergic receptor (beta(2)AR) overexpressor mice." CIRCULATION 102.18 (October 31, 2000): 94-94.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
94
End Page
94

Both beta-adrenergic receptor kinase 1 and cAMP-dependent protein kinase mediate agonist-induced beta(1)-adrenergic receptor sequestration.

Authors
Rapacciuolo, A; Cong, M; Luttrell, LM; Kohout, TA; Lefkowitz, RJ; Rockman, HA
MLA Citation
Rapacciuolo, A, Cong, M, Luttrell, LM, Kohout, TA, Lefkowitz, RJ, and Rockman, HA. "Both beta-adrenergic receptor kinase 1 and cAMP-dependent protein kinase mediate agonist-induced beta(1)-adrenergic receptor sequestration." CIRCULATION 102.18 (October 31, 2000): 28-29.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
28
End Page
29

Differential patterns of gene expression in development and rescue of mouse heart failure

Authors
Blaxall, BC; Lefkowitz, RJ; Koch, WJ
MLA Citation
Blaxall, BC, Lefkowitz, RJ, and Koch, WJ. "Differential patterns of gene expression in development and rescue of mouse heart failure." CIRCULATION 102.18 (October 31, 2000): 30-30.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
30
End Page
30

beta-arrestin2 gene deletion attenuates pressure overload hypertrophy

Authors
Feldman, DS; Esposito, G; Lin, F; Mao, L; Lefkowitz, RJ; Rockman, HA
MLA Citation
Feldman, DS, Esposito, G, Lin, F, Mao, L, Lefkowitz, RJ, and Rockman, HA. "beta-arrestin2 gene deletion attenuates pressure overload hypertrophy." CIRCULATION 102.18 (October 31, 2000): 29-29.
Source
wos-lite
Published In
Circulation
Volume
102
Issue
18
Publish Date
2000
Start Page
29
End Page
29

Reply: receptor specificity of G-protein-coupled receptor kinases

Authors
Gainetdinov, RR; Premont, RT; Caron, MG; Lefkowitz, RJ
MLA Citation
Gainetdinov, RR, Premont, RT, Caron, MG, and Lefkowitz, RJ. "Reply: receptor specificity of G-protein-coupled receptor kinases." Trends Pharmacol Sci 21.10 (October 2000): 366-367.
PMID
11050311
Source
pubmed
Published In
Trends in Pharmacological Sciences
Volume
21
Issue
10
Publish Date
2000
Start Page
366
End Page
367

Adenovirus-mediated genetic manipulation of the myocardial beta-adrenergic signaling system in transplanted hearts.

OBJECTIVES: Ex vivo perfusion of the cardiac allograft during organ procurement is an ideal environment for adenoviral vectors with transgenes that target improving graft contractility. One such target is the beta-adrenergic receptor-signaling system, in which alterations in transgenic mice have elucidated novel means to improve the function of the heart in vivo. The purpose of the current study was to determine the functional consequences of beta-adrenergic receptor manipulation in a rabbit model of cardiac allograft transplantation. METHODS: New Zealand White rabbits weighing 3 kg served as recipients to 1-kg outbred donors. Donor hearts were arrested and harvested, and 1 of 3 adenoviral constructs was administered into the aortic root perfusing the graft. Transgenes delivered encoded either the human beta(2)-adrenergic receptor, a peptide inhibitor of beta-adrenergic receptor densensitization, or the marker transgene beta-galactosidase. RESULTS: Five days after cervical heterotopic transplantation, left ventricular performance was measured on a Langendorff apparatus. A moderate pattern of rejection was seen in all grafts. Biventricular myocyte expression of beta-galactosidase was observed, and beta(2)-adrenergic receptor density was elevated 10-fold in grafts that received adeno-beta(2)-adrenergic receptor. Left ventricular systolic and diastolic performance was significantly increased in grafts transfected with either adeno-beta(2)-adrenergic receptor or adeno-beta-adrenergic receptor densensitization compared with control grafts that received adeno-beta-galactosidase. CONCLUSIONS: Ex vivo adenovirus-mediated gene transfer is feasible in a rabbit allograft model and, more important, genetic manipulation of beta-adrenergic receptor signaling either by increasing beta(2)-adrenergic receptor density or blocking endogenous receptor desensitization improves graft function acutely in this allograft model.

Authors
Shah, AS; White, DC; Tai, O; Hata, JA; Wilson, KH; Pippen, A; Kypson, AP; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Shah, AS, White, DC, Tai, O, Hata, JA, Wilson, KH, Pippen, A, Kypson, AP, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Adenovirus-mediated genetic manipulation of the myocardial beta-adrenergic signaling system in transplanted hearts." J Thorac Cardiovasc Surg 120.3 (September 2000): 581-588.
PMID
10962422
Source
pubmed
Published In
Journal of Thoracic and Cardiovascular Surgery
Volume
120
Issue
3
Publish Date
2000
Start Page
581
End Page
588

The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing.

We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of beta(2)-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.

Authors
Premont, RT; Claing, A; Vitale, N; Perry, SJ; Lefkowitz, RJ
MLA Citation
Premont, RT, Claing, A, Vitale, N, Perry, SJ, and Lefkowitz, RJ. "The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing." J Biol Chem 275.29 (July 21, 2000): 22373-22380.
PMID
10896954
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
29
Publish Date
2000
Start Page
22373
End Page
22380

The superfamily of heptahelical receptors.

Despite a growing appreciation of functional analogies between visual and hormonal signalling systems in the early 1980s, the discovery of the close structural relationship between rhodopsin and the beta2-adrenergic receptor, and of the existence of a larger 'superfamily' of such receptors, came as a total surprise. Here I provide a personal perspective on events leading up to and flowing from this exciting discovery that opened up a vast field of research.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "The superfamily of heptahelical receptors." Nat Cell Biol 2.7 (July 2000): E133-E136.
PMID
10878827
Source
pubmed
Published In
Nature Cell Biology
Volume
2
Issue
7
Publish Date
2000
Start Page
E133
End Page
E136
DOI
10.1038/35017152

alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro.

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Herein we report that alpha-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with alpha-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and alpha-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and alpha-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of alpha-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity.

Authors
Freeman, JL; Pitcher, JA; Li, X; Bennett, V; Lefkowitz, RJ
MLA Citation
Freeman, JL, Pitcher, JA, Li, X, Bennett, V, and Lefkowitz, RJ. "alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro." FEBS Lett 473.3 (May 19, 2000): 280-284.
PMID
10818226
Source
pubmed
Published In
FEBS Letters
Volume
473
Issue
3
Publish Date
2000
Start Page
280
End Page
284

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

Authors
White, DC; Hata, JA; Shah, AS; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
White, DC, Hata, JA, Shah, AS, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction." Proc Natl Acad Sci U S A 97.10 (May 9, 2000): 5428-5433.
Website
http://hdl.handle.net/10161/7811
PMID
10779554
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
10
Publish Date
2000
Start Page
5428
End Page
5433
DOI
10.1073/pnas.090091197

GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6.

ADP-ribosylation factor (ARF) proteins are key players in numerous vesicular trafficking events ranging from the formation and fusion of vesicles in the Golgi apparatus to exocytosis and endocytosis. To complete their GTPase cycle, ARFs require a guanine nucleotide-exchange protein to catalyze replacement of GDP by GTP and a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Recently numerous guanine nucleotide-exchange proteins and GAP proteins have been identified and partially characterized. Every ARF GAP protein identified to date contains a characteristic zinc finger motif. GIT1 and GIT2, two members of a new family of G protein-coupled receptor kinase-interacting proteins, also contain a putative zinc finger motif and display ARF GAP activity. Truncation of the amino-terminal region containing the zinc finger motif prevented GAP activity of GIT1. One zinc molecule was found associated per molecule of purified recombinant ARF-GAP1, GIT1, and GIT2 proteins, suggesting the zinc finger motifs of ARF GAPs are functional and should play an important role in their GAP activity. Unlike ARF-GAP1, GIT1 and GIT2 stimulate hydrolysis of GTP bound to ARF6. Accordingly we found that the phospholipid dependence of the GAP activity of ARF-GAP1 and GIT proteins was quite different, as the GIT proteins are stimulated by phosphatidylinositol 3,4, 5-trisphosphate whereas ARF-GAP1 is stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. These results suggest that although the mechanism of GTP hydrolysis is probably very similar in these two families of ARF GAPs, GIT proteins might specifically regulate the activity of ARF6 in cells in coordination with phosphatidylinositol 3-kinase signaling pathways.

Authors
Vitale, N; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ; Premont, RT
MLA Citation
Vitale, N, Patton, WA, Moss, J, Vaughan, M, Lefkowitz, RJ, and Premont, RT. "GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6." J Biol Chem 275.18 (May 5, 2000): 13901-13906.
PMID
10788515
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
18
Publish Date
2000
Start Page
13901
End Page
13906

beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis.

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.

Authors
Miller, WE; Maudsley, S; Ahn, S; Khan, KD; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Miller, WE, Maudsley, S, Ahn, S, Khan, KD, Luttrell, LM, and Lefkowitz, RJ. "beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis." J Biol Chem 275.15 (April 14, 2000): 11312-11319.
PMID
10753943
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
15
Publish Date
2000
Start Page
11312
End Page
11319

Catecholamines, cardiac beta-adrenergic receptors, and heart failure.

Authors
Lefkowitz, RJ; Rockman, HA; Koch, WJ
MLA Citation
Lefkowitz, RJ, Rockman, HA, and Koch, WJ. "Catecholamines, cardiac beta-adrenergic receptors, and heart failure." Circulation 101.14 (April 11, 2000): 1634-1637. (Review)
Website
http://hdl.handle.net/10161/5909
PMID
10758041
Source
pubmed
Published In
Circulation
Volume
101
Issue
14
Publish Date
2000
Start Page
1634
End Page
1637

The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor.

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the "transactivation" of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. beta(2)-Adrenergic receptor (beta(2)AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the beta(2)AR and the "transactivated" EGFR. beta(2)AR-mediated EGFR phosphorylation and subsequent beta(2)AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. beta(2)AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.

Authors
Maudsley, S; Pierce, KL; Zamah, AM; Miller, WE; Ahn, S; Daaka, Y; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Maudsley, S, Pierce, KL, Zamah, AM, Miller, WE, Ahn, S, Daaka, Y, Lefkowitz, RJ, and Luttrell, LM. "The beta(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor." J Biol Chem 275.13 (March 31, 2000): 9572-9580.
PMID
10734107
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
13
Publish Date
2000
Start Page
9572
End Page
9580

The β2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the 'transactivation' of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. β2- Adrenergic receptor (β2AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the β2AR and the 'transactivated' EGFR. β2AR-mediated EGFR phosphorylation and subsequent β2AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. β2AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.

Authors
Maudsley, S; Pierce, KL; Zamah, AM; Miller, WE; Ahn, S; Daaka, Y; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Maudsley, S, Pierce, KL, Zamah, AM, Miller, WE, Ahn, S, Daaka, Y, Lefkowitz, RJ, and Luttrell, LM. "The β2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor." Journal of Biological Chemistry 275.13 (March 31, 2000): 9572-9580.
Source
scopus
Published In
The Journal of biological chemistry
Volume
275
Issue
13
Publish Date
2000
Start Page
9572
End Page
9580
DOI
10.1074/jbc.275.13.9572

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors.

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Authors
Pierce, KL; Maudsley, S; Daaka, Y; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Pierce, KL, Maudsley, S, Daaka, Y, Luttrell, LM, and Lefkowitz, RJ. "Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors." Proc Natl Acad Sci U S A 97.4 (February 15, 2000): 1489-1494.
Website
http://hdl.handle.net/10161/7814
PMID
10677489
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
4
Publish Date
2000
Start Page
1489
End Page
1494

Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance.

BACKGROUND: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. METHODS AND RESULTS: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. CONCLUSIONS: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Authors
Shah, AS; Lilly, RE; Kypson, AP; Tai, O; Hata, JA; Pippen, A; Silvestry, SC; Lefkowitz, RJ; Glower, DD; Koch, WJ
MLA Citation
Shah, AS, Lilly, RE, Kypson, AP, Tai, O, Hata, JA, Pippen, A, Silvestry, SC, Lefkowitz, RJ, Glower, DD, and Koch, WJ. "Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance." Circulation 101.4 (February 1, 2000): 408-414.
Website
http://hdl.handle.net/10161/7825
PMID
10653833
Source
pubmed
Published In
Circulation
Volume
101
Issue
4
Publish Date
2000
Start Page
408
End Page
414

Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.

Authors
Claing, A; Perry, SJ; Achiriloaie, M; Walker, JK; Albanesi, JP; Lefkowitz, RJ; Premont, RT
MLA Citation
Claing, A, Perry, SJ, Achiriloaie, M, Walker, JK, Albanesi, JP, Lefkowitz, RJ, and Premont, RT. "Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity." Proc Natl Acad Sci U S A 97.3 (February 1, 2000): 1119-1124.
Website
http://hdl.handle.net/10161/7812
PMID
10655494
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
3
Publish Date
2000
Start Page
1119
End Page
1124

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Authors
Eckhart, AD; Duncan, SJ; Penn, RB; Benovic, JL; Lefkowitz, RJ; Koch, WJ
MLA Citation
Eckhart, AD, Duncan, SJ, Penn, RB, Benovic, JL, Lefkowitz, RJ, and Koch, WJ. "Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart." Circ Res 86.1 (January 7, 2000): 43-50.
Website
http://hdl.handle.net/10161/5938
PMID
10625304
Source
pubmed
Published In
Circulation Research
Volume
86
Issue
1
Publish Date
2000
Start Page
43
End Page
50

Functional consequences of altering myocardial adrenergic receptor signaling.

From the ability to successfully manipulate the mouse genome has come important transgenic and gene-targeted knockout models that impact many areas of biomedical research. Genetically engineered mouse models geared toward the study of cardiovascular regulation have recently been described and provide powerful tools to study normal and compromised cardiac physiology. The genetic manipulation of the adrenergic receptor (AR) signaling system in the heart, including its regulation by desensitizing kinases, has shed light on the role of this signaling pathway in the regulation of cardiac contractility. One major finding, supported by several mouse models, is that in vivo contractility can be enhanced via alteration of myocardial AR signaling. Thus genetic manipulation of this critical receptor system in the heart represents a novel therapeutic approach for improving function of the failing heart.

Authors
Koch, WJ; Lefkowitz, RJ; Rockman, HA
MLA Citation
Koch, WJ, Lefkowitz, RJ, and Rockman, HA. "Functional consequences of altering myocardial adrenergic receptor signaling." Annu Rev Physiol 62 (2000): 237-260. (Review)
PMID
10845091
Source
pubmed
Published In
Annual Review of Physiology
Volume
62
Publish Date
2000
Start Page
237
End Page
260
DOI
10.1146/annurev.physiol.62.1.237

β-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of β-arrestin1-dependent targeting of c-SRC in receptor endocytosis

β-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the β2-adrenergic receptor in a β-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of β-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between β-arrestin1 and the catalytic or kinase domain (SH1) of cSRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by β-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block β2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by cSRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the β-arrestin1/c-SRC interaction but also implicate β-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.

Authors
Miller, WE; Maudsley, S; Ahn, S; Khan, KD; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Miller, WE, Maudsley, S, Ahn, S, Khan, KD, Luttrell, LM, and Lefkowitz, RJ. "β-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of β-arrestin1-dependent targeting of c-SRC in receptor endocytosis." Journal of Biological Chemistry 275.15 (2000): 11312-11319.
Source
scival
Published In
Journal of Biological Chemistry
Volume
275
Issue
15
Publish Date
2000
Start Page
11312
End Page
11319
DOI
10.1074/jbc.275.15.11312

The β2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the 'transactivation' of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. β2- Adrenergic receptor (β2AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the β2AR and the 'transactivated' EGFR. β2AR-mediated EGFR phosphorylation and subsequent β2AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. β2AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.

Authors
Maudsley, S; Pierce, KL; Zamah, AM; Miller, WE; Ahn, S; Daaka, Y; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Maudsley, S, Pierce, KL, Zamah, AM, Miller, WE, Ahn, S, Daaka, Y, Lefkowitz, RJ, and Luttrell, LM. "The β2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor." Journal of Biological Chemistry 275.13 (2000): 9572-9580.
Source
scival
Published In
Journal of Biological Chemistry
Volume
275
Issue
13
Publish Date
2000
Start Page
9572
End Page
9580
DOI
10.1074/jbc.275.13.9572

α-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Herein we report that α-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with α-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and α-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and α-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of α-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity. Copyright (C) 2000 Federation of European Biochemical Societies.

Authors
Freeman, JLR; Pitcher, JA; Li, X; Bennett, V; Lefkowitz, RJ
MLA Citation
Freeman, JLR, Pitcher, JA, Li, X, Bennett, V, and Lefkowitz, RJ. "α-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro." FEBS Letters 473.3 (2000): 280-284.
Source
scival
Published In
FEBS Letters
Volume
473
Issue
3
Publish Date
2000
Start Page
280
End Page
284
DOI
10.1016/S0014-5793(00)01543-X

β-Arrestin 2: A receptor-regulated MAPK scaffold for the activation of JNK3

β-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of β-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfectecl COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with β-arrestin 2. Cellular transfection of β-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of β-arrestin 2 and active JNK3 to intracellular vesicles. Thus, β-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Authors
McDonald, PH; Chow, C-W; Miller, WE; Laporte, SA; Field, ME; Lin, F-T; Davis, RJ; Lefkowitz, RJ
MLA Citation
McDonald, PH, Chow, C-W, Miller, WE, Laporte, SA, Field, ME, Lin, F-T, Davis, RJ, and Lefkowitz, RJ. "β-Arrestin 2: A receptor-regulated MAPK scaffold for the activation of JNK3." Science 290.5496 (2000): 1574-1577.
Source
scival
Published In
Science
Volume
290
Issue
5496
Publish Date
2000
Start Page
1574
End Page
1577

Inhibition of spontaneous β2-adrenergic activation rescues β1- adrenergic contractile response in cardiomyocytes overexpressing β2- adrenoceptor

Cardiac-specific overexpression of the human β2-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand- independent spontaneous β2-AR activation. However, agonist-mediated stimulation of either β1-AR or β2-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of β2-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to β1-AR stimulation by norepinephrine and an α1-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since β-adrenergic receptor kinase, βARK1, activity remains unaltered, the unresponsiveness of β1-AR is not caused by βARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10-5 M), or a β2-AR inverse agonist, ICI 118,551 (5 x 10-7 M), to abolish spontaneous β2-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the β1-AR contractile response. The 'rescued' contractile response is completely reversed by a β1-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human β2-AR results in elevated baseline cAMP and contraction associated with a marked attenuation of β1-AR response; carbachol pretreatment fully revives the diminished β1-AR contractile response. Thus, we conclude that constitutive β2-AR activation induces a heterologous desensitization of β1-ARs independent of βARK1 and G(i) proteins; suppression of the constitutive β2-AR signaling by either a β2-AR inverse agonist or stimulation of the muscarinic receptor rescues the β1-ARs from desensitization, permitting agonist-induced contractile response.

Authors
Zhang, S-J; Cheng, H; Zhou, Y-Y; Wang, D-J; Zhu, W; Ziman, B; Spurgoen, H; Lefkowitz, RJ; Lakatta, EG; Koch, WJ; Xiao, R-P
MLA Citation
Zhang, S-J, Cheng, H, Zhou, Y-Y, Wang, D-J, Zhu, W, Ziman, B, Spurgoen, H, Lefkowitz, RJ, Lakatta, EG, Koch, WJ, and Xiao, R-P. "Inhibition of spontaneous β2-adrenergic activation rescues β1- adrenergic contractile response in cardiomyocytes overexpressing β2- adrenoceptor." Journal of Biological Chemistry 275.28 (2000): 21773-21779.
PMID
10787424
Source
scival
Published In
Journal of Biological Chemistry
Volume
275
Issue
28
Publish Date
2000
Start Page
21773
End Page
21779
DOI
10.1074/jbc.M909484199

Assembly of an A kinase-anchoring protein-β2-adrenergic receptor complex facilitates receptor phosphorylation and signaling

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the β2-adrenergic receptor (β2-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4,5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the β2-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the β2-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the β2-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance β2-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.

Authors
Fraser, IDC; Cong, M; Kim, J; Rollins, EN; Daaka, Y; Lefkowitz, RJ; Scott, JD
MLA Citation
Fraser, IDC, Cong, M, Kim, J, Rollins, EN, Daaka, Y, Lefkowitz, RJ, and Scott, JD. "Assembly of an A kinase-anchoring protein-β2-adrenergic receptor complex facilitates receptor phosphorylation and signaling." Current Biology 10.7 (2000): 409-412.
PMID
10753752
Source
scival
Published In
Current Biology
Volume
10
Issue
7
Publish Date
2000
Start Page
409
End Page
412
DOI
10.1016/S0960-9822(00)00419-X

Enhanced morphine analgesia in mice lacking beta-arrestin 2.

The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.

Authors
Bohn, LM; Lefkowitz, RJ; Gainetdinov, RR; Peppel, K; Caron, MG; Lin, FT
MLA Citation
Bohn, LM, Lefkowitz, RJ, Gainetdinov, RR, Peppel, K, Caron, MG, and Lin, FT. "Enhanced morphine analgesia in mice lacking beta-arrestin 2." Science 286.5449 (December 24, 1999): 2495-2498.
PMID
10617462
Source
pubmed
Published In
Science
Volume
286
Issue
5449
Publish Date
1999
Start Page
2495
End Page
2498

Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases.

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.

Authors
Pitcher, JA; Tesmer, JJ; Freeman, JL; Capel, WD; Stone, WC; Lefkowitz, RJ
MLA Citation
Pitcher, JA, Tesmer, JJ, Freeman, JL, Capel, WD, Stone, WC, and Lefkowitz, RJ. "Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases." J Biol Chem 274.49 (December 3, 1999): 34531-34534.
PMID
10574913
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
49
Publish Date
1999
Start Page
34531
End Page
34534

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

Authors
Gainetdinov, RR; Bohn, LM; Walker, JK; Laporte, SA; Macrae, AD; Caron, MG; Lefkowitz, RJ; Premont, RT
MLA Citation
Gainetdinov, RR, Bohn, LM, Walker, JK, Laporte, SA, Macrae, AD, Caron, MG, Lefkowitz, RJ, and Premont, RT. "Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice." Neuron 24.4 (December 1999): 1029-1036.
PMID
10624964
Source
pubmed
Published In
Neuron
Volume
24
Issue
4
Publish Date
1999
Start Page
1029
End Page
1036

Expression of beta-adrenergic receptor kinase 1 inhibitor fails to prevent the development or progression of heart failure in CREBA133 transgenic mice

Authors
Fentzke, RC; Lin, H; Korcarz, CE; Lang, RM; Koch, W; Lefkowitz, RJ; Leiden, JM; Leiden, JM
MLA Citation
Fentzke, RC, Lin, H, Korcarz, CE, Lang, RM, Koch, W, Lefkowitz, RJ, Leiden, JM, and Leiden, JM. "Expression of beta-adrenergic receptor kinase 1 inhibitor fails to prevent the development or progression of heart failure in CREBA133 transgenic mice." CIRCULATION 100.18 (November 2, 1999): 494-494.
Source
wos-lite
Published In
Circulation
Volume
100
Issue
18
Publish Date
1999
Start Page
494
End Page
494

Expression of a beta ARK-1 inhibitor increases myocardial contractility without increasing susceptibility to ischemic injury

Authors
Cross, HR; Steenbergen, C; Lefkowitz, RJ; Murphy, E
MLA Citation
Cross, HR, Steenbergen, C, Lefkowitz, RJ, and Murphy, E. "Expression of a beta ARK-1 inhibitor increases myocardial contractility without increasing susceptibility to ischemic injury." CIRCULATION 100.18 (November 2, 1999): 493-493.
Source
wos-lite
Published In
Circulation
Volume
100
Issue
18
Publish Date
1999
Start Page
493
End Page
493

Muscarinic stimulation rescues beta(1)-adrenoceptors from desensitization induced by constitutively active beta(2)-adrenoceptors in cardiac myocytes

Authors
Xiao, RP; Cheng, HP; Zhou, YY; Wang, DG; Lefkowitz, RJ; Zhang, SJ; Koch, WJ; Lakatta, EG
MLA Citation
Xiao, RP, Cheng, HP, Zhou, YY, Wang, DG, Lefkowitz, RJ, Zhang, SJ, Koch, WJ, and Lakatta, EG. "Muscarinic stimulation rescues beta(1)-adrenoceptors from desensitization induced by constitutively active beta(2)-adrenoceptors in cardiac myocytes." CIRCULATION 100.18 (November 2, 1999): 488-488.
Source
wos-lite
Published In
Circulation
Volume
100
Issue
18
Publish Date
1999
Start Page
488
End Page
488

beta ARK1 inhibition improves survival and cardiac function in a mouse model of severe cardiomyopathy

Authors
Harding, VB; Rapacciuolo, A; Mao, L; Lefkowitz, RJ; Rockman, HA
MLA Citation
Harding, VB, Rapacciuolo, A, Mao, L, Lefkowitz, RJ, and Rockman, HA. "beta ARK1 inhibition improves survival and cardiac function in a mouse model of severe cardiomyopathy." CIRCULATION 100.18 (November 2, 1999): 552-552.
Source
wos-lite
Published In
Circulation
Volume
100
Issue
18
Publish Date
1999
Start Page
552
End Page
552

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Authors
Tang, Y; Hu, LA; Miller, WE; Ringstad, N; Hall, RA; Pitcher, JA; DeCamilli, P; Lefkowitz, RJ
MLA Citation
Tang, Y, Hu, LA, Miller, WE, Ringstad, N, Hall, RA, Pitcher, JA, DeCamilli, P, and Lefkowitz, RJ. "Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor." Proc Natl Acad Sci U S A 96.22 (October 26, 1999): 12559-12564.
Website
http://hdl.handle.net/10161/7817
PMID
10535961
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
22
Publish Date
1999
Start Page
12559
End Page
12564

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.

Authors
Premont, RT; Macrae, AD; Aparicio, SA; Kendall, HE; Welch, JE; Lefkowitz, RJ
MLA Citation
Premont, RT, Macrae, AD, Aparicio, SA, Kendall, HE, Welch, JE, and Lefkowitz, RJ. "The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation." J Biol Chem 274.41 (October 8, 1999): 29381-29389.
PMID
10506199
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
41
Publish Date
1999
Start Page
29381
End Page
29389

Myocardial G protein-coupled receptor kinases: implications for heart failure therapy.

The beta-adrenergic signaling cascade is an important regulator of myocardial function. Significant alterations of this pathway are associated with several cardiovascular diseases, including congestive heart failure (CHF). Included in these alterations is increased activity and expression of G protein-coupled receptor kinases (GRKs), such as the beta-adrenergic receptor kinase (beta ARK1), which phosphorylate and desensitize beta-adrenergic receptors (beta ARs). A body of evidence is accumulating that suggests that GRKs, in particular beta ARK1, are critical determinants of cardiac function under normal conditions and in disease states. Transgenic mice with myocardial-targeted alterations of GRK activity have shown profound changes in the in vivo functional performance of the heart. Included in these studies is the compelling finding that inhibition of beta ARK1 activity or expression significantly enhances cardiac function and potentiates beta AR signaling in failing cardiomyocytes. This article summarizes the advances made in the study of beta ARK1 in the heart and addresses its potential as a novel therapeutic target for CHF.

Authors
Iaccarino, G; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Lefkowitz, RJ, and Koch, WJ. "Myocardial G protein-coupled receptor kinases: implications for heart failure therapy." Proc Assoc Am Physicians 111.5 (September 1999): 399-405. (Review)
PMID
10519160
Source
pubmed
Published In
Proceedings of the Association of American Physicians
Volume
111
Issue
5
Publish Date
1999
Start Page
399
End Page
405

G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction.

The Na(+)/H(+) exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.

Authors
Hall, RA; Spurney, RF; Premont, RT; Rahman, N; Blitzer, JT; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Hall, RA, Spurney, RF, Premont, RT, Rahman, N, Blitzer, JT, Pitcher, JA, and Lefkowitz, RJ. "G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction." J Biol Chem 274.34 (August 20, 1999): 24328-24334.
PMID
10446210
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
34
Publish Date
1999
Start Page
24328
End Page
24334

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

Authors
Akhter, SA; Eckhart, AD; Rockman, HA; Shotwell, K; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Eckhart, AD, Rockman, HA, Shotwell, K, Lefkowitz, RJ, and Koch, WJ. "In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function." Circulation 100.6 (August 10, 1999): 648-653.
Website
http://hdl.handle.net/10161/5907
PMID
10441103
Source
pubmed
Published In
Circulation
Volume
100
Issue
6
Publish Date
1999
Start Page
648
End Page
653

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Authors
Maurice, JP; Hata, JA; Shah, AS; White, DC; McDonald, PH; Dolber, PC; Wilson, KH; Lefkowitz, RJ; Glower, DD; Koch, WJ
MLA Citation
Maurice, JP, Hata, JA, Shah, AS, White, DC, McDonald, PH, Dolber, PC, Wilson, KH, Lefkowitz, RJ, Glower, DD, and Koch, WJ. "Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery." J Clin Invest 104.1 (July 1999): 21-29.
Website
http://hdl.handle.net/10161/5923
PMID
10393695
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
104
Issue
1
Publish Date
1999
Start Page
21
End Page
29
DOI
10.1172/JCI6026

Feedback regulation of beta-arrestin1 function by extracellular signal-regulated kinases.

The functions of beta-arrestin1 to facilitate clathrin-mediated endocytosis of the beta2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic beta-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of beta-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that beta-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate beta-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates beta-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated beta-arrestin1. Under such conditions, beta-arrestin1-mediated beta2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated beta-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate beta-arrestin1, thereby exerting an inhibitory feedback control of its function.

Authors
Lin, FT; Miller, WE; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Lin, FT, Miller, WE, Luttrell, LM, and Lefkowitz, RJ. "Feedback regulation of beta-arrestin1 function by extracellular signal-regulated kinases." J Biol Chem 274.23 (June 4, 1999): 15971-15974.
PMID
10347142
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
23
Publish Date
1999
Start Page
15971
End Page
15974

Heptahelical receptor signaling: beyond the G protein paradigm.

Authors
Hall, RA; Premont, RT; Lefkowitz, RJ
MLA Citation
Hall, RA, Premont, RT, and Lefkowitz, RJ. "Heptahelical receptor signaling: beyond the G protein paradigm." J Cell Biol 145.5 (May 31, 1999): 927-932. (Review)
Website
http://hdl.handle.net/10161/5922
PMID
10352011
Source
pubmed
Published In
The Journal of Cell Biology
Volume
145
Issue
5
Publish Date
1999
Start Page
927
End Page
932

Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases.

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.

Authors
Della Rocca, GJ; Maudsley, S; Daaka, Y; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Della Rocca, GJ, Maudsley, S, Daaka, Y, Lefkowitz, RJ, and Luttrell, LM. "Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases." The Journal of biological chemistry 274.20 (May 1999): 13978-13984.
PMID
10318809
Source
epmc
Published In
The Journal of biological chemistry
Volume
274
Issue
20
Publish Date
1999
Start Page
13978
End Page
13984
DOI
10.1074/jbc.274.20.13978

Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation.

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

Authors
McDonald, PH; Cote, NL; Lin, FT; Premont, RT; Pitcher, JA; Lefkowitz, RJ
MLA Citation
McDonald, PH, Cote, NL, Lin, FT, Premont, RT, Pitcher, JA, and Lefkowitz, RJ. "Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation." J Biol Chem 274.16 (April 16, 1999): 10677-10680.
PMID
10196135
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
16
Publish Date
1999
Start Page
10677
End Page
10680

beta(2)-adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels

Authors
An, RH; Heath, BM; Higgins, JP; Koch, WJ; Lefkowitz, RJ; Kass, RS
MLA Citation
An, RH, Heath, BM, Higgins, JP, Koch, WJ, Lefkowitz, RJ, and Kass, RS. "beta(2)-adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels." JOURNAL OF PHYSIOLOGY-LONDON 516.1 (April 1, 1999): 19-30.
PMID
10066919
Source
wos-lite
Published In
The Journal of Physiology
Volume
516
Issue
1
Publish Date
1999
Start Page
19
End Page
30
DOI
10.1111/j.1469-7793.1999.019aa.x

Altered airway and cardiac responses in mice lacking G protein-coupled receptor kinase 3.

Contraction and relaxation of airway smooth muscles is mediated, in part, by G protein-coupled receptors (GPCRs) and dysfunction of these receptors has been implicated in asthma. Phosphorylation of GPCRs, by G protein-coupled receptor kinase (GRK), is an important mechanism involved in the dampening of GPCR signaling. To determine whether this mechanism might play a role in airway smooth muscle physiology, we examined the airway pressure time index and heart rate (HR) responses to intravenous administration of the cholinergic agonist methacholine (MCh) in genetically altered mice lacking one copy of GRK2 (GRK2 +/-), homozygous GRK3 knockout (GRK3 -/-), and wild-type littermates. (GRK2 -/- mice die in utero.) GRK3 -/- mice demonstrated a significant enhancement in the airway response to 100 and 250 microgram/kg doses of MCh compared with wild-type and GRK2 +/- mice. GRK3 -/- mice also displayed an enhanced sensitivity of the airway smooth muscle response to MCh. In addition, GRK3 -/- mice displayed an altered HR recovery from MCh-induced bradycardia. Although direct stimulation of cardiac muscarinic receptors measured as vagal stimulation-induced bradycardia was similar in GRK3 -/- and wild-type mice, the baroreflex increase in HR associated with sodium nitroprusside-induced hypotension was significantly greater in GRK3 -/- than wild-type mice. Therefore, these data demonstrate that in the mouse, GRK3 may be involved in modulating the cholinergic response of airway smooth muscle and in regulating the chronotropic component of the baroreceptor reflex.

Authors
Walker, JK; Peppel, K; Lefkowitz, RJ; Caron, MG; Fisher, JT
MLA Citation
Walker, JK, Peppel, K, Lefkowitz, RJ, Caron, MG, and Fisher, JT. "Altered airway and cardiac responses in mice lacking G protein-coupled receptor kinase 3." Am J Physiol 276.4 Pt 2 (April 1999): R1214-R1221.
PMID
10198406
Source
pubmed
Published In
The American journal of physiology
Volume
276
Issue
4 Pt 2
Publish Date
1999
Start Page
R1214
End Page
R1221

Regulation of tyrosine kinase cascades by G-protein-coupled receptors.

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.

Authors
Luttrell, LM; Daaka, Y; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Daaka, Y, and Lefkowitz, RJ. "Regulation of tyrosine kinase cascades by G-protein-coupled receptors." Curr Opin Cell Biol 11.2 (April 1999): 177-183. (Review)
PMID
10209148
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
11
Issue
2
Publish Date
1999
Start Page
177
End Page
183

Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis.

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Authors
Iaccarino, G; Smithwick, LA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Smithwick, LA, Lefkowitz, RJ, and Koch, WJ. "Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis." Proc Natl Acad Sci U S A 96.7 (March 30, 1999): 3945-3950.
Website
http://hdl.handle.net/10161/7819
PMID
10097143
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
7
Publish Date
1999
Start Page
3945
End Page
3950

Altered airway response in mice lacking G protein-coupled receptor kinase 3 (GRK3)

Authors
Walker, JKL; Peppel, K; Lefkowitz, RJ; Caron, MG; Fisher, JT
MLA Citation
Walker, JKL, Peppel, K, Lefkowitz, RJ, Caron, MG, and Fisher, JT. "Altered airway response in mice lacking G protein-coupled receptor kinase 3 (GRK3)." FASEB JOURNAL 13.4 (March 12, 1999): A169-A169.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
13
Issue
4
Publish Date
1999
Start Page
A169
End Page
A169

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Authors
Della Rocca, GJ; Mukhin, YV; Garnovskaya, MN; Daaka, Y; Clark, GJ; Luttrell, LM; Lefkowitz, RJ; Raymond, JR
MLA Citation
Della Rocca, GJ, Mukhin, YV, Garnovskaya, MN, Daaka, Y, Clark, GJ, Luttrell, LM, Lefkowitz, RJ, and Raymond, JR. "Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis." The Journal of biological chemistry 274.8 (February 1999): 4749-4753.
PMID
9988712
Source
epmc
Published In
The Journal of biological chemistry
Volume
274
Issue
8
Publish Date
1999
Start Page
4749
End Page
4753
DOI
10.1074/jbc.274.8.4749

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Authors
Luttrell, LM; Ferguson, SS; Daaka, Y; Miller, WE; Maudsley, S; Della Rocca, GJ; Lin, F; Kawakatsu, H; Owada, K; Luttrell, DK; Caron, MG; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Ferguson, SS, Daaka, Y, Miller, WE, Maudsley, S, Della Rocca, GJ, Lin, F, Kawakatsu, H, Owada, K, Luttrell, DK, Caron, MG, and Lefkowitz, RJ. "Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes." Science 283.5402 (January 29, 1999): 655-661.
PMID
9924018
Source
pubmed
Published In
Science
Volume
283
Issue
5402
Publish Date
1999
Start Page
655
End Page
661

β-Arrestin-Dependent Formation of β 2 Adrenergic Receptor-Src Protein Kinase Complexes

Authors
Luttrell, LM; Ferguson, SSG; Daaka, Y; Miller, WE; Maudsley, S; Della Rocca, GJ; Lin, F-T; Kawakatsu, H; Owada, K; Luttrell, DK; Caron, MG; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Ferguson, SSG, Daaka, Y, Miller, WE, Maudsley, S, Della Rocca, GJ, Lin, F-T, Kawakatsu, H, Owada, K, Luttrell, DK, Caron, MG, and Lefkowitz, RJ. "β-Arrestin-Dependent Formation of β 2 Adrenergic Receptor-Src Protein Kinase Complexes ." Science 283.5402 (January 29, 1999): 655-661.
Source
crossref
Published In
Science
Volume
283
Issue
5402
Publish Date
1999
Start Page
655
End Page
661
DOI
10.1126/science.283.5402.655

Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling.

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.

Authors
Ahn, S; Maudsley, S; Luttrell, LM; Lefkowitz, RJ; Daaka, Y
MLA Citation
Ahn, S, Maudsley, S, Luttrell, LM, Lefkowitz, RJ, and Daaka, Y. "Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling." J Biol Chem 274.3 (January 15, 1999): 1185-1188.
PMID
9880482
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
3
Publish Date
1999
Start Page
1185
End Page
1188

Bbeta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: regulation by beta- but not alpha1-adrenergic stimulation.

Pressure overload ventricular hypertrophy is accompanied by dysfunctional beta-adrenergic receptor signaling due to increased levels of the beta-adrenergic receptor kinase-1, which phosphorylates and desensitizes beta-adrenergic receptors. In this study, we examined whether increased beta-adrenergic receptor kinase 1 expression is associated with myocardial hypertrophy induced by adrenergic stimulation. With use of implanted mini-osmotic pumps, we treated mice with isoproterenol, phenylephrine, or vehicle to distinguish between alpha1- and beta-adrenergic stimulation. Both treatments resulted in cardiac hypertrophy, but only isoproterenol induced significant increases in beta-adrenergic receptor kinase-1 protein levels and activity. Similarly, in isolated adult rat cardiac myocytes, 24 hours of isoproterenol stimulation resulted in a significant 2.8-fold increase in beta-adrenergic receptor kinase-1 protein levels, whereas 24 hours of phenylephrine treatment did not alter beta-adrenergic receptor kinase-1 expression. Our results indicate that increased beta-adrenergic receptor kinase-1 is not invariably associated with myocardial hypertrophy but apparently is controlled by the state of beta-adrenergic receptor activation.

Authors
Iaccarino, G; Dolber, PC; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Dolber, PC, Lefkowitz, RJ, and Koch, WJ. "Bbeta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: regulation by beta- but not alpha1-adrenergic stimulation." Hypertension 33.1 Pt 2 (January 1999): 396-401.
Website
http://hdl.handle.net/10161/7815
PMID
9931136
Source
pubmed
Published In
Hypertension
Volume
33
Issue
1 Pt 2
Publish Date
1999
Start Page
396
End Page
401

beta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy - Regulation by beta- but not alpha(1)-adrenergic stimulation

Authors
Iaccarino, G; Dolber, PC; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Dolber, PC, Lefkowitz, RJ, and Koch, WJ. "beta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy - Regulation by beta- but not alpha(1)-adrenergic stimulation." January 1999.
Source
wos-lite
Published In
Hypertension
Volume
33
Issue
1
Publish Date
1999
Start Page
396
End Page
401

Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of β2-adrenergic receptors (β2-ARs) leads to significantly enhanced cardiac function. Tuus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human β2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the β2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the β2-AR agonist, zinterol. Treatment with the β2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean β-AR density which corresponded to a significant increase in basal contractility (LV + dP/dt(max), control: 3152.1 ± 286 versus β2-AR, 6250.6* ± 432.5 mmHg/s; n = 10, *P < 0.02). β2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of β2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the postoperative setting.

Authors
Kypson, AP; Hendrickson, SC; Akhter, SA; Wilson, K; McDonald, PH; Lilly, RE; Dolber, PC; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Kypson, AP, Hendrickson, SC, Akhter, SA, Wilson, K, McDonald, PH, Lilly, RE, Dolber, PC, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function." Gene Therapy 6.7 (1999): 1298-1304.
PMID
10455439
Source
scival
Published In
Gene Therapy
Volume
6
Issue
7
Publish Date
1999
Start Page
1298
End Page
1304
DOI
10.1038/sj.gt.3300940

β-Adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: Regulation by β- but not α1-adrenergic stimulation

Pressure overload ventricular hypertrophy is accompanied by dysfunctional β-adrenergic receptor signaling due to increased levels of the β-adrenergic receptor kinase-1, which phosphorylates and desensitizes β- adrenergic receptors. In this study, we examined whether increased β- adrenergic receptor kinase 1 expression is associated with myocardial hypertrophy induced by adrenergic stimulation. With use of implanted mini- osmotic pumps, we treated mice with isoproterenol, phenylephrine, or vehicle to distinguish between α1- and β-adrenergic stimulation. Both treatments resulted in cardiac hypertrophy, but only isoproterenol induced significant increases in β-adrenergic receptor kinase-1 protein levels and activity. Similarly, in isolated adult rat cardiac myocytes, 24 hours of isoproterenol stimulation resulted in a significant 2.8-fold increase in β-adrenergic receptor kinase-1 protein levels, whereas 24 hours of phenylephrine treatment did not alter β-adrenergic receptor kinase-1 expression. Our results indicate that increased β-adrenergic receptor kinase-1 is not invariably associated with myocardial hypertrophy but apparently is controlled by the state of β-adrenergic receptor activation.

Authors
Iaccarino, G; Dolber, PC; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Dolber, PC, Lefkowitz, RJ, and Koch, WJ. "β-Adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: Regulation by β- but not α1-adrenergic stimulation." Hypertension 33.1 II (1999): 396-401.
Source
scival
Published In
Hypertension
Volume
33
Issue
1 II
Publish Date
1999
Start Page
396
End Page
401

β-Arrestins regulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth factor I receptor

β-Arrestins mediate agonist-dependent desensitization of G protein- coupled receptors and target the receptors to clathrin-coated pits for internalization. Here we report an expanded role of β-arrestins in promoting clathrin-mediated endocytosis of a tyrosine kinase growth factor receptor, i.e. the insulin-like growth factor I (IGF-1) receptor. β-Arrestins bind to the ligand-occupied IGF-1 receptors, promote their endocytosis, and enhance IGF-1-dependent mitogen-activated protein kinase phosphorylation and DNA synthesis. Our results suggest a role for β-arrestins in regulating mitogenic signaling and clathrin-mediated endocytosis of receptors not classically coupled to G proteins.

Authors
Lin, F-T; Daaka, Y; Lefkowitz, RJ
MLA Citation
Lin, F-T, Daaka, Y, and Lefkowitz, RJ. "β-Arrestins regulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth factor I receptor." Journal of Biological Chemistry 273.48 (1999): 31640-31643.
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
48
Publish Date
1999
Start Page
31640
End Page
31643
DOI
10.1074/jbc.273.48.31640

Overexpression of the cardiac β2-adrenergic receptor and expression of a β-adrenergic receptor kinase-1 (βARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury

Cardiac β2-adrenergic receptor (β2AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing β2ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of β2AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20- minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because β2ARs, unlike β1ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TD4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either βARK1 (TGβARK1) or a βARK1 inhibitor (TGβARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGβARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGβARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, β2AR overexpression increased ischemic injury, whereas βARK1 overexpression was protective. Ischemic injury in the β2AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike β2AR overexpression, basal contractility was increased by βARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.

Authors
Cross, HR; Steenbergen, C; Lefkowitz, RJ; Koch, WJ; Murphy, E
MLA Citation
Cross, HR, Steenbergen, C, Lefkowitz, RJ, Koch, WJ, and Murphy, E. "Overexpression of the cardiac β2-adrenergic receptor and expression of a β-adrenergic receptor kinase-1 (βARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury." Circulation Research 85.11 (1999): 1077-1084.
Website
http://hdl.handle.net/10161/7818
PMID
10571539
Source
scival
Published In
Circulation Research
Volume
85
Issue
11
Publish Date
1999
Start Page
1077
End Page
1084

Hetahelical receptor signaling: Beyond the G protein paradigm

Authors
Hall, RA; Premont, RT; Lefkowitz, RJ
MLA Citation
Hall, RA, Premont, RT, and Lefkowitz, RJ. "Hetahelical receptor signaling: Beyond the G protein paradigm." Journal of Cell Biology 145.5 (1999): 927-932.
Website
http://hdl.handle.net/10161/7816
Source
scival
Published In
Journal of Cell Biology
Volume
145
Issue
5
Publish Date
1999
Start Page
927
End Page
932
DOI
10.1083/jcb.145.5.927

Beta2-adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels.

1. We studied the effect of overexpression of the beta2-adrenergic receptor (beta2-AR) in the heart on ion channel currents in single cells isolated from hearts of fetal and neonatal transgenic and wild-type mice. The beta2-AR transgene construct was under the control of the murine alpha-myosin heavy chain (alpha-MHC) promoter, and ion channel activity was measured at distinct developmental stages using whole-cell and perforated patch clamp techniques. 2. We found no change in L-type Ca2+ channel current (ICa) density in early embryonic stages (E11-13) of beta2-AR transgenic positive (TG+) mice, but significant increases in ICa density in intermediate (E14-16, 152 %) and late (E17-19, 173.7 %) fetal and neonatal (1 day post partum, 161 %) TG+ compared with transgenic negative (TG-) mice. This increase in ICa was accompanied by a negative shift in the peak of the current-voltage relationship in TG+ mice. 3. Transient (< 3 min) or prolonged (16-24 h) exposure of TG- neonatal stage myocytes to 8-Br-cAMP (300 microM) increased ICa density and caused a shift in the current-voltage relationship to a similar extent to that seen in TG+ mice. In TG+ myocytes, 8-Br-cAMP had no effect. Exposure of TG+ cells to Rp-cAMPS reversed both the shift in voltage dependence and reduced the peak current density observed in these myocytes. We concluded from these results that the L-type Ca2+ channel is maximally modulated by cAMP-dependent protein kinase (PKA) in TG+ mice and that the alpha-MHC promoter is functional in the ventricle as early as embryonic day 14. 4. In contrast, we found that slow delayed rectifier K+ channels were not changed significantly at any of the developmental stages studied by the overexpression of beta2-ARs compared with TG- mice. The sensitivity of murine slow delayed rectifier K+ channels to cAMP was tested by both transient and prolonged exposure to 8-Br-cAMP (300 microM), which increased the slow delayed rectifier K+ channel current (IK,s) density to a similar extent in both TG- and TG+ neonatal myocytes. In addition, we found that there was no difference in the concentration dependence of the response of ICa and IK,s to 8-Br-cAMP. 5. Thus, overexpression of the beta2-AR in the heart results in distinct modulation of ICa, but not IK,s, and this is not due to differences in the 8-Br-cAMP sensitivity of the two channels. Instead, these results are consistent with both compartmentalization of beta2-AR-controlled cAMP and distinct localization of L-type Ca2+ and slow delayed rectifier K+ channels. This cAMP is targeted preferentially to the L-type Ca2+ channel and is not accessible to the slow delayed rectifier K+ channel.

Authors
An, R; Heath, BM; Higgins, JP; Koch, WJ; Lefkowitz, RJ; Kass, RS
MLA Citation
An, R, Heath, BM, Higgins, JP, Koch, WJ, Lefkowitz, RJ, and Kass, RS. "Beta2-adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels." The Journal of physiology 516 (1999): Pt 1/--.
Source
scival
Published In
The Journal of Physiology
Volume
516
Publish Date
1999
Start Page
Pt 1/-
DOI
10.1111/j.1469-7793.1999.019aa.x

Coupling of β2-adrenoceptor to G(i) proteins and its physiological relevance in murine cardiac myocytes

Transgenic mouse models have been developed to manipulate β-adrenergic receptor (βAR) signal transduction. Although several of these models have altered βAR subtypes, the specific functional sequelae of βAR stimulation in murine heart, particularly those of β2-adrenergic receptor (β2AR) stimulation, have not been characterized. In the present study, we investigated effects of β2AR stimulation on contraction, [Ca2+](i) transient, and L-type Ca2+ currents (I(Ca)) in single ventricular myocytes isolated from transgenic mice overexpressing human β2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous β2AR activation. In contrast, β2AR stimulation by zinterol or isoproterenol plus a selective β1-adrenergic receptor (β1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, β2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the I(Ca), [Ca2+](i), and contractile responses to β2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac β2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist- induced β2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [γ-32P]GTP azidoanilide into α subunits of G(i2) and G(i3) after β2AR stimulation by zinterol or isoproterenol plus the β1AR blocker CGP 20712A. This effect to activate G(i) proteins was abolished by a selective β2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) β2ARs in murine cardiac myocytes couple to concurrent G(s) and G(i) signaling, resulting in null inotropic response, unless the G(i) signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to β2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of β2AR-coupled G(i) proteins; and (3) spontaneous β2AR activation may differ from agonist-stimulated β2AR signaling.

Authors
Xiao, R-P; Avdonin, P; Zhou, Y-Y; Cheng, H; Akhter, SA; Eschenhagen, T; Lefkowitz, RJ; Koch, WJ; Lakatta, EG
MLA Citation
Xiao, R-P, Avdonin, P, Zhou, Y-Y, Cheng, H, Akhter, SA, Eschenhagen, T, Lefkowitz, RJ, Koch, WJ, and Lakatta, EG. "Coupling of β2-adrenoceptor to G(i) proteins and its physiological relevance in murine cardiac myocytes." Circulation Research 84.1 (1999): 43-52.
Website
http://hdl.handle.net/10161/5912
PMID
9915773
Source
scival
Published In
Circulation Research
Volume
84
Issue
1
Publish Date
1999
Start Page
43
End Page
52

beta-arrestins regulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth factor I receptor.

beta-Arrestins mediate agonist-dependent desensitization of G protein-coupled receptors and target the receptors to clathrin-coated pits for internalization. Here we report an expanded role of beta-arrestins in promoting clathrin-mediated endocytosis of a tyrosine kinase growth factor receptor, i.e. the insulin-like growth factor I (IGF-1) receptor. beta-Arrestins bind to the ligand-occupied IGF-1 receptors, promote their endocytosis, and enhance IGF-1-dependent mitogen-activated protein kinase phosphorylation and DNA synthesis. Our results suggest a role for beta-arrestins in regulating mitogenic signaling and clathrin-mediated endocytosis of receptors not classically coupled to G proteins.

Authors
Lin, FT; Daaka, Y; Lefkowitz, RJ
MLA Citation
Lin, FT, Daaka, Y, and Lefkowitz, RJ. "beta-arrestins regulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth factor I receptor." J Biol Chem 273.48 (November 27, 1998): 31640-31643.
PMID
9822622
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
48
Publish Date
1998
Start Page
31640
End Page
31643

beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Authors
Premont, RT; Claing, A; Vitale, N; Freeman, JL; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
MLA Citation
Premont, RT, Claing, A, Vitale, N, Freeman, JL, Pitcher, JA, Patton, WA, Moss, J, Vaughan, M, and Lefkowitz, RJ. "beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein." Proc Natl Acad Sci U S A 95.24 (November 24, 1998): 14082-14087.
Website
http://hdl.handle.net/10161/7826
PMID
9826657
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
24
Publish Date
1998
Start Page
14082
End Page
14087

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase- activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2- adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Authors
Premont, RT; Claing, A; Vitale, N; Freeman, JLR; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
MLA Citation
Premont, RT, Claing, A, Vitale, N, Freeman, JLR, Pitcher, JA, Patton, WA, Moss, J, Vaughan, M, and Lefkowitz, RJ. 2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein." Proceedings of the National Academy of Sciences of the United States of America 95.24 (November 24, 1998): 14082-14087.
Source
scopus
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
24
Publish Date
1998
Start Page
14082
End Page
14087
DOI
10.1073/pnas.95.24.14082

Palmitoylation increases the kinase activity of the G protein-coupled receptor kinase, GRK6.

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Authors
Stoffel, RH; Inglese, J; Macrae, AD; Lefkowitz, RJ; Premont, RT
MLA Citation
Stoffel, RH, Inglese, J, Macrae, AD, Lefkowitz, RJ, and Premont, RT. "Palmitoylation increases the kinase activity of the G protein-coupled receptor kinase, GRK6." Biochemistry 37.46 (November 17, 1998): 16053-16059.
PMID
9819198
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
46
Publish Date
1998
Start Page
16053
End Page
16059
DOI
10.1021/bi981432d

Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by beta-adrenergic receptor stimulation and blockade.

BACKGROUND: Impaired myocardial beta-adrenergic receptor (betaAR) signaling, including desensitization and functional uncoupling, is a characteristic of congestive heart failure. A contributing mechanism for this impairment may involve enhanced myocardial beta-adrenergic receptor kinase (betaARK1) activity because levels of this betaAR-desensitizing G protein-coupled receptor kinase (GRK) are increased in heart failure. An hypothesis has emerged that increased sympathetic nervous system activity associated with heart failure might be the initial stimulus for betaAR signaling alterations, including desensitization. We have chronically treated mice with drugs that either activate or antagonize betaARs to study the dynamic relationship between betaAR activation and myocardial levels of betaARK1. METHODS AND RESULTS: Long-term in vivo stimulation of betaARs results in the impairment of cardiac +betaAR signaling and increases the level of expression (mRNA and protein) and activity of +betaARK1 but not that of GRK5, a second GRK abundantly expressed in the myocardium. Long-term beta-blocker treatment, including the use of carvedilol, improves myocardial betaAR signaling and reduces betaARK1 levels in a specific and dose-dependent manner. Identical results were obtained in vitro in cultured cells, demonstrating that the regulation of GRK expression is directly linked to betaAR signaling. CONCLUSIONS: This report demonstrates, for the first time, that betaAR stimulation can significantly increase the expression of betaARK1 , whereas beta-blockade decreases expression. This reciprocal regulation of betaARK1 documents a novel mechanism of ligand-induced betaAR regulation and provides important insights into the potential mechanisms responsible for the effectiveness of beta-blockers, such as carvedilol, in the treatment of heart failure.

Authors
Iaccarino, G; Tomhave, ED; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Tomhave, ED, Lefkowitz, RJ, and Koch, WJ. "Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by beta-adrenergic receptor stimulation and blockade." Circulation 98.17 (October 27, 1998): 1783-1789.
Website
http://hdl.handle.net/10161/5902
PMID
9788834
Source
pubmed
Published In
Circulation
Volume
98
Issue
17
Publish Date
1998
Start Page
1783
End Page
1789

Global myocardial overpression of beta(2)-adrenergic receptors via intracoronary adenovirus delivery enhances in vivo contractility

Authors
Maurice, JP; Hata, JA; Shah, AS; Wilson, KH; McDonald, PH; Lefkowitz, RJ; Glower, DD; Koch, WJ
MLA Citation
Maurice, JP, Hata, JA, Shah, AS, Wilson, KH, McDonald, PH, Lefkowitz, RJ, Glower, DD, and Koch, WJ. "Global myocardial overpression of beta(2)-adrenergic receptors via intracoronary adenovirus delivery enhances in vivo contractility." October 27, 1998.
Source
wos-lite
Published In
Circulation
Volume
98
Issue
17
Publish Date
1998
Start Page
737
End Page
737

Increased mortality and cardiac beta ARK1 levels in mice with cardiac overexpression of the alpha 1b adrenergic receptor chronically treated with phenylephrine.

Authors
Iaccarino, G; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Lefkowitz, RJ, and Koch, WJ. "Increased mortality and cardiac beta ARK1 levels in mice with cardiac overexpression of the alpha 1b adrenergic receptor chronically treated with phenylephrine." CIRCULATION 98.17 (October 27, 1998): 69-69.
Source
wos-lite
Published In
Circulation
Volume
98
Issue
17
Publish Date
1998
Start Page
69
End Page
69

G(beta g) inhibition attenuates vascular smooth muscle cell proliferation in vitro and in vivo

Authors
Iaccarino, G; Smithwick, A; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Smithwick, A, Lefkowitz, RJ, and Koch, WJ. "G(beta g) inhibition attenuates vascular smooth muscle cell proliferation in vitro and in vivo." CIRCULATION 98.17 (October 27, 1998): 740-740.
Source
wos-lite
Published In
Circulation
Volume
98
Issue
17
Publish Date
1998
Start Page
740
End Page
740

beta adrenergic receptor kinase 1 (beta ARK1) expression in catecholamine induced cardiac hypertrophy

Authors
Iaccarino, G; Trimarco, B; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Trimarco, B, Lefkowitz, RJ, and Koch, WJ. "beta adrenergic receptor kinase 1 (beta ARK1) expression in catecholamine induced cardiac hypertrophy." HYPERTENSION 32.3 (September 1998): 600-600.
Source
wos-lite
Published In
Hypertension
Volume
32
Issue
3
Publish Date
1998
Start Page
600
End Page
600

Longitudinal evaluation of the myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF/Mcc-fa(C)P) rats

Authors
Anderson, KM; Eckhart, AD; Willette, RN; Lefkowitz, RJ; Koch, WJ
MLA Citation
Anderson, KM, Eckhart, AD, Willette, RN, Lefkowitz, RJ, and Koch, WJ. "Longitudinal evaluation of the myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF/Mcc-fa(C)P) rats." HYPERTENSION 32.3 (September 1998): 631-631.
Source
wos-lite
Published In
Hypertension
Volume
32
Issue
3
Publish Date
1998
Start Page
631
End Page
631

Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin.

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806-3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.

Authors
Freeman, JL; De La Cruz, EM; Pollard, TD; Lefkowitz, RJ; Pitcher, JA
MLA Citation
Freeman, JL, De La Cruz, EM, Pollard, TD, Lefkowitz, RJ, and Pitcher, JA. "Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin." J Biol Chem 273.32 (August 7, 1998): 20653-20657.
PMID
9685424
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
32
Publish Date
1998
Start Page
20653
End Page
20657

G protein signaling and vein graft intimal hyperplasia: reduction of intimal hyperplasia in vein grafts by a Gbetagamma inhibitor suggests a major role of G protein signaling in lesion development.

Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.

Authors
Davies, MG; Huynh, TT; Fulton, GJ; Lefkowitz, RJ; Svendsen, E; Hagen, PO; Koch, WJ
MLA Citation
Davies, MG, Huynh, TT, Fulton, GJ, Lefkowitz, RJ, Svendsen, E, Hagen, PO, and Koch, WJ. "G protein signaling and vein graft intimal hyperplasia: reduction of intimal hyperplasia in vein grafts by a Gbetagamma inhibitor suggests a major role of G protein signaling in lesion development." Arterioscler Thromb Vasc Biol 18.8 (August 1998): 1275-1280.
Website
http://hdl.handle.net/10161/7824
PMID
9714134
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
18
Issue
8
Publish Date
1998
Start Page
1275
End Page
1280

G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization." J Biol Chem 273.30 (July 24, 1998): 18677-18680. (Review)
PMID
9668034
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
30
Publish Date
1998
Start Page
18677
End Page
18680

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

Authors
Hall, RA; Ostedgaard, LS; Premont, RT; Blitzer, JT; Rahman, N; Welsh, MJ; Lefkowitz, RJ
MLA Citation
Hall, RA, Ostedgaard, LS, Premont, RT, Blitzer, JT, Rahman, N, Welsh, MJ, and Lefkowitz, RJ. "A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins." Proc Natl Acad Sci U S A 95.15 (July 21, 1998): 8496-8501.
Website
http://hdl.handle.net/10161/7821
PMID
9671706
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
15
Publish Date
1998
Start Page
8496
End Page
8501

Control of myocardial contractile function by the level of beta-adrenergic receptor kinase 1 in gene-targeted mice.

We studied the effect of alterations in the level of myocardial beta-adrenergic receptor kinase betaARK1) in two types of genetically altered mice. The first group is heterozygous for betaARK1 gene ablation, betaARK1(+/-), and the second is not only heterozygous for betaARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a betaARK1 COOH-terminal inhibitor peptide, betaARK1(+/-)betaARKct. In contrast to the embryonic lethal phenotype of the homozygous betaARK1 knockout (Jaber, M., Koch, W. J., Rockman, H. A., Smith, B., Bond, R. A., Sulik, K., Ross, J., Jr., Lefkowitz, R. J., Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), betaARK1(+/-) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the betaARK1(+/-) and betaARK1(+/-)betaARKct mice with the greatest level observed in the betaARK1(+/-)betaARKct animals. Contractile parameters were measured in adult myocytes isolated from both groups of gene-targeted animals. A significantly greater increase in percent cell shortening and rate of cell shortening following isoproterenol stimulation was observed in the betaARK1(+/-) and betaARK1(+/-)betaARKct myocytes compared with wild-type cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of betaARK1 activity. This has important implications in disease states such as heart failure (in which betaARK1 activity is increased) and suggests that betaARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of betaARK1 activity enhances beta-adrenergic receptor signaling leading to improved functional catecholamine responsiveness.

Authors
Rockman, HA; Choi, DJ; Akhter, SA; Jaber, M; Giros, B; Lefkowitz, RJ; Caron, MG; Koch, WJ
MLA Citation
Rockman, HA, Choi, DJ, Akhter, SA, Jaber, M, Giros, B, Lefkowitz, RJ, Caron, MG, and Koch, WJ. "Control of myocardial contractile function by the level of beta-adrenergic receptor kinase 1 in gene-targeted mice." J Biol Chem 273.29 (July 17, 1998): 18180-18184.
PMID
9660778
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
29
Publish Date
1998
Start Page
18180
End Page
18184

Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.

Heart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR were mated into a genetic model of murine heart failure (MLP-/-). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP-/- and MLP-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR desensitization in the MLP-/- mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the betaARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.

Authors
Rockman, HA; Chien, KR; Choi, DJ; Iaccarino, G; Hunter, JJ; Ross, J; Lefkowitz, RJ; Koch, WJ
MLA Citation
Rockman, HA, Chien, KR, Choi, DJ, Iaccarino, G, Hunter, JJ, Ross, J, Lefkowitz, RJ, and Koch, WJ. "Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice." Proc Natl Acad Sci U S A 95.12 (June 9, 1998): 7000-7005.
Website
http://hdl.handle.net/10161/7822
PMID
9618528
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
12
Publish Date
1998
Start Page
7000
End Page
7005

The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin.

The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.

Authors
Pitcher, JA; Hall, RA; Daaka, Y; Zhang, J; Ferguson, SS; Hester, S; Miller, S; Caron, MG; Lefkowitz, RJ; Barak, LS
MLA Citation
Pitcher, JA, Hall, RA, Daaka, Y, Zhang, J, Ferguson, SS, Hester, S, Miller, S, Caron, MG, Lefkowitz, RJ, and Barak, LS. "The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin." J Biol Chem 273.20 (May 15, 1998): 12316-12324.
PMID
9575184
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
20
Publish Date
1998
Start Page
12316
End Page
12324

Enhanced vasorelaxation by overexpression of beta 2-adrenergic receptors in large arteries.

This study was designed to determine if adenoviral-mediated delivery of a transgene encoding the beta 2-adrenergic receptor (beta 2-AR) to the carotid arterial wall could result in alterations in in vivo vascular function. De-endothelialized rat carotid arteries were infused in vivo with 0.1 mg/ml elastase and adenovirus [6 x 10(9) plaque forming units (PFU)] containing either the marker gene beta-galactosidase (Adeno-beta-gal), DNA encoding the human beta 2-AR (Adeno-beta 2-AR), or no transgene. This low concentration of elastase increased the water permeability (5.2 +/- 0.6 v 1.9 +/- 0.4 x 10(-8) cm/s/mmHg, n = 4, P < 0.0001) without affecting either the vasomotor responsiveness or the morphology of the arterial wall. A transfection efficiency of 73% was achieved with Adeno-beta-gal (n = 3). beta-gal expression was associated with infrequent appearance of T and B lymphocytes, or neutrophil infiltration. Five days after infection with Adeno-beta 2-AR, the total beta-AR density increased six-fold (67.8 +/- 3.4 v 397.0 +/- 155.5 fmol/mg protein, n = 5, P < 0.01); isoproterenol-induced vasorelaxation at transmural pressures from 10-110/mmHg increased (P < 0.01) compared to arteries exposed to control virus (empty adenovirus), n = 4; and isoproterenol-stimulated cAMP production was increased by 65% (n = 5). Thus, adenoviral-mediated delivery of beta 2-ARs into large artery walls results in enhanced beta-AR-mediated vasorelaxation via augmentation in cAMP levels in vascular smooth muscle cells.

Authors
Gaballa, MA; Peppel, K; Lefkowitz, RJ; Aguirre, M; Dolber, PC; Pennock, GD; Koch, WJ; Goldman, S
MLA Citation
Gaballa, MA, Peppel, K, Lefkowitz, RJ, Aguirre, M, Dolber, PC, Pennock, GD, Koch, WJ, and Goldman, S. "Enhanced vasorelaxation by overexpression of beta 2-adrenergic receptors in large arteries." J Mol Cell Cardiol 30.5 (May 1998): 1037-1045.
PMID
9618244
Source
pubmed
Published In
Journal of Molecular and Cellular Cardiology
Volume
30
Issue
5
Publish Date
1998
Start Page
1037
End Page
1045
DOI
10.1006/jmcc.1998.0668

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Authors
Akhter, SA; Luttrell, LM; Rockman, HA; Iaccarino, G; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Luttrell, LM, Rockman, HA, Iaccarino, G, Lefkowitz, RJ, and Koch, WJ. "Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy." Science 280.5363 (April 24, 1998): 574-577.
PMID
9554846
Source
pubmed
Published In
Science
Volume
280
Issue
5363
Publish Date
1998
Start Page
574
End Page
577

The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange.

Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

Authors
Hall, RA; Premont, RT; Chow, CW; Blitzer, JT; Pitcher, JA; Claing, A; Stoffel, RH; Barak, LS; Shenolikar, S; Weinman, EJ; Grinstein, S; Lefkowitz, RJ
MLA Citation
Hall, RA, Premont, RT, Chow, CW, Blitzer, JT, Pitcher, JA, Claing, A, Stoffel, RH, Barak, LS, Shenolikar, S, Weinman, EJ, Grinstein, S, and Lefkowitz, RJ. "The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange." Nature 392.6676 (April 9, 1998): 626-630.
PMID
9560162
Source
pubmed
Published In
Nature
Volume
392
Issue
6676
Publish Date
1998
Start Page
626
End Page
630
DOI
10.1038/33458

The β2-adrenergic receptor interacts with the Na(+/H+)-exchanger regulatory factor to control Na(+/H+) exchange

Stimulation of β2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of β(2)-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the β2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the β2- adrenergic receptor by means of a PDZdomain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the β2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters β2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent β2-adrenergic receptor binding of NHERF play a role in β2adrenergic receptor-mediated regulation of Na+/H+ exchange.

Authors
Hall, RA; Premont, RT; Chow, CW; Blitzer, JT; Pitcher, JA; Claing, A; Stoffel, RH; Barak, LS; Shenolikar, S; Weinman, EJ; Grinstein, S; Lefkowitz, RJ
MLA Citation
Hall, RA, Premont, RT, Chow, CW, Blitzer, JT, Pitcher, JA, Claing, A, Stoffel, RH, Barak, LS, Shenolikar, S, Weinman, EJ, Grinstein, S, and Lefkowitz, RJ. "The β2-adrenergic receptor interacts with the Na(+/H+)-exchanger regulatory factor to control Na(+/H+) exchange." Nature 392.6676 (April 9, 1998): 626-630.
Source
scopus
Published In
Nature
Volume
392
Issue
6676
Publish Date
1998
Start Page
626
End Page
630
DOI
10.1038/33458

Ex vivo adenovirus-mediated gene transfer to the adult rat heart.

OBJECTIVE: The ability to transfer genes to adult myocardium may have therapeutic implications for cardiac transplantation. We investigated the feasibility of adenovirus-mediated transfer of marker genes LacZ and Luciferase, as well as the potentially therapeutic gene of the human beta2-adrenergic receptor in a rat heterotopic heart transplant model. METHODS: Donor hearts were flushed with 10(12) total viral particles of one of three transgenes. Hearts were harvested at various time points after transplantation. LacZ-treated hearts were assessed by histologic staining and Luciferase-treated hearts were assayed for specific luminescence activity. Hearts treated with beta2-adrenergic receptor underwent radioligand binding assays and immunohistochemistry with the use of an antibody specific for the human beta2-adrenergic receptor. RESULTS: LacZ hearts revealed diffuse myocyte staining as opposed to none within controls at 5 days. Luciferase hearts demonstrated a mean activity of 970,000 +/- 220,000 arbitrary light units versus 500 +/- 200 for the controls (p = 0.001). Total beta2-adrenergic receptor densities (fmol/mg membrane protein) for hearts that received the beta2-adrenergic receptor transgene at 3, 5, 7, 10, and 14 days after infection were as follows: right ventricle, 488.5 +/- 126.8, 519.4 +/- 81.8,* 477.1 +/- 51.8,* 183.0 +/- 6.5,* and 82.7 +/- 19.1; left ventricle, 511.0 +/- 167.6, 1206.4 +/- 321.8,* 525.3 +/- 188.7, 183.5 +/- 18.6,* and 75.9 +/- 15.2 (*p < 0.05 vs control value of 75.6 +/- 6.4). Immunohistochemical analysis revealed diffuse staining of varying intensity within myocardial sarcolemmal membranes. CONCLUSIONS: We conclude that global overexpression of different transgenes is possible during cardiac transplantation and, ultimately, adenovirus-mediated gene transfer may provide a unique opportunity for genetic manipulation of the donor organ, potentially enhancing its function.

Authors
Kypson, AP; Peppel, K; Akhter, SA; Lilly, RE; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Kypson, AP, Peppel, K, Akhter, SA, Lilly, RE, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Ex vivo adenovirus-mediated gene transfer to the adult rat heart." J Thorac Cardiovasc Surg 115.3 (March 1998): 623-630.
PMID
9535450
Source
pubmed
Published In
Journal of Thoracic and Cardiovascular Surgery
Volume
115
Issue
3
Publish Date
1998
Start Page
623
End Page
630
DOI
10.1016/S0022-5223(98)70327-7

Targeting of cAMP-dependent increases in L-type Ca+2 (I-Ca) over delayed K+ channel (I-Ks) activity by overexpression of the beta(2)-adrenergic receptor (beta(2)AR) in the developing mouse heart.

Authors
Heath, B; An, RH; Koch, W; Lefkowitz, RJ; Kass, RS
MLA Citation
Heath, B, An, RH, Koch, W, Lefkowitz, RJ, and Kass, RS. "Targeting of cAMP-dependent increases in L-type Ca+2 (I-Ca) over delayed K+ channel (I-Ks) activity by overexpression of the beta(2)-adrenergic receptor (beta(2)AR) in the developing mouse heart." BIOPHYSICAL JOURNAL 74.2 (February 1998): A35-A35.
Source
wos-lite
Published In
Biophysical Journal
Volume
74
Issue
2
Publish Date
1998
Start Page
A35
End Page
A35

Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase.

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and beta-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of beta-arrestin or dynamin, beta2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and beta-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.

Authors
Daaka, Y; Luttrell, LM; Ahn, S; Della Rocca, GJ; Ferguson, SS; Caron, MG; Lefkowitz, RJ
MLA Citation
Daaka, Y, Luttrell, LM, Ahn, S, Della Rocca, GJ, Ferguson, SS, Caron, MG, and Lefkowitz, RJ. "Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase." The Journal of biological chemistry 273.2 (January 1998): 685-688.
PMID
9422717
Source
epmc
Published In
The Journal of biological chemistry
Volume
273
Issue
2
Publish Date
1998
Start Page
685
End Page
688
DOI
10.1074/jbc.273.2.685

Regulation of mitogen-activated protein kinase pathways by catecholamine receptors.

Authors
Luttrell, LM; van Biesen, T; Hawes, BE; Della Rocca, GJ; Luttrell, DK; Lefkowitz, RJ
MLA Citation
Luttrell, LM, van Biesen, T, Hawes, BE, Della Rocca, GJ, Luttrell, DK, and Lefkowitz, RJ. "Regulation of mitogen-activated protein kinase pathways by catecholamine receptors." Advances in pharmacology (San Diego, Calif.) 42 (January 1998): 466-470.
PMID
9327940
Source
epmc
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Publish Date
1998
Start Page
466
End Page
470

Myocardial overexpression of adrenergic receptors and receptor kinases.

Authors
Koch, WJ; Lefkowitz, RJ; Milano, CA; Akhter, SA; Rockman, HA
MLA Citation
Koch, WJ, Lefkowitz, RJ, Milano, CA, Akhter, SA, and Rockman, HA. "Myocardial overexpression of adrenergic receptors and receptor kinases." Adv Pharmacol 42 (1998): 502-506.
PMID
9327950
Source
pubmed
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Publish Date
1998
Start Page
502
End Page
506

G protein-coupled receptor kinases.

G protein-coupled receptor kinases (GRKs) constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) as their primary substrates. GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization. This review focuses on the regulation of GRK activity by a variety of allosteric and other factors: agonist-stimulated GPCRs, beta gamma subunits of heterotrimeric GTP-binding proteins, phospholipid cofactors, the calcium-binding proteins calmodulin and recoverin, posttranslational isoprenylation and palmitoylation, autophosphorylation, and protein kinase C-mediated GRK phosphorylation. Studies employing recombinant, purified proteins, cell culture, and transgenic animal models attest to the general importance of GRKs in regulating a vast array of GPCRs both in vitro and in vivo.

Authors
Pitcher, JA; Freedman, NJ; Lefkowitz, RJ
MLA Citation
Pitcher, JA, Freedman, NJ, and Lefkowitz, RJ. "G protein-coupled receptor kinases." Annu Rev Biochem 67 (1998): 653-692. (Review)
PMID
9759500
Source
pubmed
Published In
Annual Review of Biochemistry
Volume
67
Publish Date
1998
Start Page
653
End Page
692
DOI
10.1146/annurev.biochem.67.1.653

Altered cardiac chronotropic responses in βARK 2 knockout mice

G protein-coupled receptor kinases (GRKs) desensitize agonist-activated G protein-coupled receptors (GPCRs) thus attenuating their responses. In cell culture experiments, GRK 3 (βARK 2) phosphorylates and desensitizes multiple GPCRs, including muscarinic acetylcholine receptors (mAChR). Genetically altered mice, lacking βARK 2 (βARK 2 -/-), display a more rapid heart rate recovery after an i.v. injection of methacholine, than wildtype mice (WT). Since this observation is contrary to that expected (assuming βARK 2 desensitizes cardiac m2AChR in vivo) and given that i.v. methacholine induces hypotension, we hypothesized that BARK 2 may desensitize a central component of cardiovascular control, the baroreceptor reflex. To test this hypothesis we examined baroreflexes as well as the impact of direct cardiac m2AChR activation with vagal stimulation. During vagal nerve stimulation (10, 15 and 20 Hz) heart rate decreased, and recovered, similarly in βARK 2 -/- and WT mice. However, the increase in heart rate associated with sodium nitroprusside-induced hypotension was significantly greater in βARK 2 -/- (88 ± 12 beats/min) than WT (31 ± 16 beats/min) mice. Interestingly, propranolol significantly decreased resting heart rate in WT (486 ± 28 to 419 ± 22 beats/min), but not βARK 2 -/-mice (443 ±26 to 426 ± 25 beats/min). In conclusion, the cardiac component of the baroreceptor reflex is likely responsible for the chronotropic differences observed between βARK 2 -/- and WT mice.

Authors
Walker, JKL; Peppel, K; Lefkowitz, RJ; Caron, MG; Fisher, JT
MLA Citation
Walker, JKL, Peppel, K, Lefkowitz, RJ, Caron, MG, and Fisher, JT. "Altered cardiac chronotropic responses in βARK 2 knockout mice." FASEB Journal 12.4 (1998): A396-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
12
Issue
4
Publish Date
1998
Start Page
A396

GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg]ZO-1) domain

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein α subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down- regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/DIg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for G(i) class α subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

Authors
Snow, BE; Hall, RA; Krumins, AM; Brothers, GM; Bouchard, D; Brothers, CA; Chung, S; Mangion, J; Gilman, AG; Lefkowitz, RJ; Siderovski, DP
MLA Citation
Snow, BE, Hall, RA, Krumins, AM, Brothers, GM, Bouchard, D, Brothers, CA, Chung, S, Mangion, J, Gilman, AG, Lefkowitz, RJ, and Siderovski, DP. "GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg]ZO-1) domain." Journal of Biological Chemistry 273.28 (1998): 17749-17755.
PMID
9651375
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
28
Publish Date
1998
Start Page
17749
End Page
17755
DOI
10.1074/jbc.273.28.17749

Molecular basis for interactions of G protein βΓ subunits with effectors

Both the α and βΓ subunits of heterotrimeric guanine nucleotide- binding proteins (G proteins) communicate signals from receptors to effectors. GβΓ subunits can regulate a diverse array of effectors, including ion channels and enzymes. Gα subunits bound to guanine diphosphate (Gα-GDP) inhibit signal transduction through GβΓ subunits, suggesting a common interface on GβΓ subunits for Gα binding and effector interaction. The molecular basis for interaction of GβΓ with effectors was characterized by mutational analysis of Gβ residues that make contact with Gα-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-β2, and β- adrenergic receptor kinase revealed the Gβ residues required for activation of each effector and provides evidence for partially overlapping domains on Gβ for regulation of these effectors. This organization of interaction regions on Gβ for different effectors and Gα explains why subunit dissociation is crucial for signal transmission through GβΓ subunits.

Authors
Ford, CE; Skiba, NP; Bae, H; Daaka, Y; Reuveny, E; Shekter, LR; Rosal, R; Weng, G; Yang, C-S; Iyengar, R; Miller, RJ; Jan, LY; Lefkowitz, RJ; Hamm, HE
MLA Citation
Ford, CE, Skiba, NP, Bae, H, Daaka, Y, Reuveny, E, Shekter, LR, Rosal, R, Weng, G, Yang, C-S, Iyengar, R, Miller, RJ, Jan, LY, Lefkowitz, RJ, and Hamm, HE. "Molecular basis for interactions of G protein βΓ subunits with effectors." Science 280.5367 (1998): 1271-1274.
PMID
9596582
Source
scival
Published In
Science
Volume
280
Issue
5367
Publish Date
1998
Start Page
1271
End Page
1274
DOI
10.1126/science.280.5367.1271

Mutagenesis of acidic residues in the β2-adrenergic receptor abolishes the requirement of low pH for receptor dephosphorylation

We have previously shown that acidic pH activates β2-adrenergic receptor (B2AR) dephosphorylation both in vitro as well as in cells, where sequestered B2ARs are localized to endosomes. To further define the role of acidic pH in receptor dephosphorylation, vesicles containing phosphorylated B2AR and bacteriorhodopsin were incubated with PP-2A. Upon illumination, an acidic environment is created inside the vesicles due to the ability of bacteriorhodopsin to act as a light-driven proton pump. While little dephosphorylation of phosphorylated B2AR was observed when the reaction was performed in the dark, illumination resulted in a 10-fold stimulation of the amount of B2AR dephosphorylated. Since only the intravesicular side of the receptor was exposed to the low pH environment, these data confirm a low pH-induced conformational change within the receptor. As acidic residues have been reported to play a role in conformational changes elicited by the low pH found in endosomes, we mutated 3 acidic residues located in the second extracellular loop of the B2AR to Ala. Compared to wild type (WT) B2AR, mutant receptor displayed an increased rate of resensitization, a process which requires receptor dephosphorylation. Further, while WT B2AR was not dephosphorylated upon incubation with phosphatase at neutral pH but only at acidic pH, the mutant B2AR was dephosphorylated at neutral pH. These data suggest that the conformation of the mutant mimics that of the WT receptor in endosomes and underscore the involvement of acidic residues in the pH-induced conformational change in the receptor.

Authors
Krueger, KM; Claing, A; Lefkowitz, RJ
MLA Citation
Krueger, KM, Claing, A, and Lefkowitz, RJ. "Mutagenesis of acidic residues in the β2-adrenergic receptor abolishes the requirement of low pH for receptor dephosphorylation." FASEB Journal 12.5 (1998): A742-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
12
Issue
5
Publish Date
1998
Start Page
A742

Mechanisms of beta-adrenergic receptor desensitization and resensitization.

Authors
Lefkowitz, RJ; Pitcher, J; Krueger, K; Daaka, Y
MLA Citation
Lefkowitz, RJ, Pitcher, J, Krueger, K, and Daaka, Y. "Mechanisms of beta-adrenergic receptor desensitization and resensitization." Adv Pharmacol 42 (1998): 416-420.
PMID
9327928
Source
pubmed
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Publish Date
1998
Start Page
416
End Page
420

New paradigms for G protein-coupled receptor signaling and desensitization.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "New paradigms for G protein-coupled receptor signaling and desensitization." NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY 358.1 (1998): R7-R7.
Source
wos-lite
Published In
Naunyn-Schmiedeberg's Archives of Pharmacology
Volume
358
Issue
1
Publish Date
1998
Start Page
R7
End Page
R7

Clathrin-mediated endocytosis of the beta-adrenergic receptor is regulated by phosphorylation/dephosphorylation of beta-arrestin1.

beta-Arrestins serve a dual regulatory role in the life cycle of G protein-coupled receptors such as the beta2-adrenergic receptor. First, they mediate rapid desensitization by binding to G protein-coupled receptor kinase-phosphorylated receptors. Second, they target the receptors for internalization into endosomal vesicles, wherein receptor dephosphorylation and resensitization occur. Here we report that phosphorylation of a carboxyl-terminal serine (Ser-412) in beta-arrestin1 regulates its endocytotic but not its desensitization function. Cytoplasmic beta-arrestin1 is constitutively phosphorylated and is recruited to the plasma membrane by agonist stimulation of the receptors. At the plasma membrane, beta-arrestin1 is rapidly dephosphorylated, a process that is required for its clathrin binding and receptor endocytosis but not for its receptor binding and desensitization. Once internalized, beta-arrestin1 is rephosphorylated. Thus, as with the classical endocytic adaptor protein complex AP2, beta-arrestin1 functions as a clathrin adaptor in receptor endocytosis which is regulated by dephosphorylation at the plasma membrane.

Authors
Lin, FT; Krueger, KM; Kendall, HE; Daaka, Y; Fredericks, ZL; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Lin, FT, Krueger, KM, Kendall, HE, Daaka, Y, Fredericks, ZL, Pitcher, JA, and Lefkowitz, RJ. "Clathrin-mediated endocytosis of the beta-adrenergic receptor is regulated by phosphorylation/dephosphorylation of beta-arrestin1." J Biol Chem 272.49 (December 5, 1997): 31051-31057.
PMID
9388255
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
49
Publish Date
1997
Start Page
31051
End Page
31057

Regulation of Mitogen-Activated Protein Kinase Pathways by Catecholamine Receptors

Authors
Luttrell, LM; Biesen, TV; Hawes, BE; Della Rocca, GJ; Luttrell, DK; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Biesen, TV, Hawes, BE, Della Rocca, GJ, Luttrell, DK, and Lefkowitz, RJ. "Regulation of Mitogen-Activated Protein Kinase Pathways by Catecholamine Receptors." Advances in Pharmacology 42.C (December 1, 1997): 466-470.
Source
scopus
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Issue
C
Publish Date
1997
Start Page
466
End Page
470
DOI
10.1016/S1054-3589(08)60789-9

G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases.

The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.

Authors
Luttrell, LM; Daaka, Y; Della Rocca, GJ; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Daaka, Y, Della Rocca, GJ, and Lefkowitz, RJ. "G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases." The Journal of biological chemistry 272.50 (December 1997): 31648-31656.
PMID
9395506
Source
epmc
Published In
The Journal of biological chemistry
Volume
272
Issue
50
Publish Date
1997
Start Page
31648
End Page
31656
DOI
10.1074/jbc.272.50.31648

Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A.

Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from Gs to Gi and initiate a new set of signalling events.

Authors
Daaka, Y; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Daaka, Y, Luttrell, LM, and Lefkowitz, RJ. "Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A." Nature 390.6655 (November 6, 1997): 88-91.
PMID
9363896
Source
pubmed
Published In
Nature
Volume
390
Issue
6655
Publish Date
1997
Start Page
88
End Page
91
DOI
10.1038/36362

Restoration of beta-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer.

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring beta-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular beta-adrenergic signaling defects including down-regulation of myocardial beta-adrenergic receptors (beta-ARs), functional beta-AR uncoupling, and an up-regulation of the beta-AR kinase (betaARK1). Adenoviral-mediated gene transfer of the human beta2-AR or an inhibitor of betaARK1 to these failing myocytes led to the restoration of beta-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of betaARK1 activity in the heart.

Authors
Akhter, SA; Skaer, CA; Kypson, AP; McDonald, PH; Peppel, KC; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Skaer, CA, Kypson, AP, McDonald, PH, Peppel, KC, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Restoration of beta-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer." Proc Natl Acad Sci U S A 94.22 (October 28, 1997): 12100-12105.
Website
http://hdl.handle.net/10161/7827
PMID
9342369
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
22
Publish Date
1997
Start Page
12100
End Page
12105

Level of beta ARK1 regulates myocardial contractile function in gene targeted mice

Authors
Choi, DJ; Koch, WJ; Rockman, HA; Akhter, SA; Lefkowitz, RJ; Caron, MG
MLA Citation
Choi, DJ, Koch, WJ, Rockman, HA, Akhter, SA, Lefkowitz, RJ, and Caron, MG. "Level of beta ARK1 regulates myocardial contractile function in gene targeted mice." CIRCULATION 96.8 (October 21, 1997): 1632-1632.
Source
wos-lite
Published In
Circulation
Volume
96
Issue
8
Publish Date
1997
Start Page
1632
End Page
1632

Adenoviral-mediated gene transfer of the beta(2)-adrenergic receptor improves function of the transplanted rat heart

Authors
Kypson, AP; Akhter, SA; Peppel, K; Hendrickson, SC; McDonald, P; Lilly, RE; Glower, DD; Lefkowitz, RJ; Koch, WJ
MLA Citation
Kypson, AP, Akhter, SA, Peppel, K, Hendrickson, SC, McDonald, P, Lilly, RE, Glower, DD, Lefkowitz, RJ, and Koch, WJ. "Adenoviral-mediated gene transfer of the beta(2)-adrenergic receptor improves function of the transplanted rat heart." October 21, 1997.
Source
wos-lite
Published In
Circulation
Volume
96
Issue
8
Publish Date
1997
Start Page
3744
End Page
3744

Reciprocal regulation of myocardial beta ARK1 by beta adrenergic agonists and antagonists

Authors
Iaccarino, G; Lefkowitz, RJ; Koch, WJ
MLA Citation
Iaccarino, G, Lefkowitz, RJ, and Koch, WJ. "Reciprocal regulation of myocardial beta ARK1 by beta adrenergic agonists and antagonists." CIRCULATION 96.8 (October 21, 1997): 2773-2773.
Source
wos-lite
Published In
Circulation
Volume
96
Issue
8
Publish Date
1997
Start Page
2773
End Page
2773

Attenuation of pressure overload hypertrophy in transgenic mice expressing an inhibitor of G(q)-coupled signaling

Authors
Akhter, SA; Luttrell, LM; Rockman, HA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Luttrell, LM, Rockman, HA, Lefkowitz, RJ, and Koch, WJ. "Attenuation of pressure overload hypertrophy in transgenic mice expressing an inhibitor of G(q)-coupled signaling." CIRCULATION 96.8 (October 21, 1997): 1635-1635.
Source
wos-lite
Published In
Circulation
Volume
96
Issue
8
Publish Date
1997
Start Page
1635
End Page
1635

G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization.

G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.

Authors
Peppel, K; Boekhoff, I; McDonald, P; Breer, H; Caron, MG; Lefkowitz, RJ
MLA Citation
Peppel, K, Boekhoff, I, McDonald, P, Breer, H, Caron, MG, and Lefkowitz, RJ. "G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization." J Biol Chem 272.41 (October 10, 1997): 25425-25428.
PMID
9325250
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
41
Publish Date
1997
Start Page
25425
End Page
25428

Transgenic mice with cardiac overexpression of alpha1B-adrenergic receptors. In vivo alpha1-adrenergic receptor-mediated regulation of beta-adrenergic signaling.

Transgenic mice were generated with cardiac-specific overexpression of the wild-type (WT) alpha1B-adrenergic receptor (AR) using the murine alpha-myosin heavy chain gene promoter. Previously, we described transgenic mice with alpha-myosin heavy chain-directed expression of a constitutively active mutant alpha1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113). In animals with >40-fold WT alpha1-AR overexpression, basal myocardial diacylglycerol content was significantly increased, indicating enhanced alpha1-adrenergic signaling and phospholipase C activity. In contrast to the mice overexpressing constitutively active mutant alpha1B-ARs, the hearts of these mice did not develop cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic factor. In vivo physiology was studied in anesthetized intact animals and showed left ventricular contractility in response to the beta-agonist isoproterenol to be significantly depressed in animals overexpressing WT alpha1B-ARs. Membranes purified from the hearts of WT alpha1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with isoproterenol, norepinephrine, or phenylephrine. Interestingly, these in vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high levels of WT alpha1B-ARs can lead to coupling to pertussis toxin-sensitive G proteins. Another potential contributor to the observed decreased myocardial signaling and function could be enhanced beta-AR desensitization as beta-adrenergic receptor kinase (betaARK1) activity was found to be significantly elevated (>3-fold) in myocardial extracts isolated from WT alpha1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart failure where betaARK1 levels are elevated and beta-ARs are down-regulated, leading to a higher percentage of cardiac alpha1-ARs. Thus, these mice serve as a unique experimental model to study the in vivo interactions between alpha- and beta-ARs in the heart.

Authors
Akhter, SA; Milano, CA; Shotwell, KF; Cho, MC; Rockman, HA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Milano, CA, Shotwell, KF, Cho, MC, Rockman, HA, Lefkowitz, RJ, and Koch, WJ. "Transgenic mice with cardiac overexpression of alpha1B-adrenergic receptors. In vivo alpha1-adrenergic receptor-mediated regulation of beta-adrenergic signaling." J Biol Chem 272.34 (August 22, 1997): 21253-21259.
PMID
9261135
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
34
Publish Date
1997
Start Page
21253
End Page
21259

Ras-dependent mitogen-activated protein kinase activation by G protein- coupled receptors

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either αIB- or α2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the α2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gβγ subunit complex sequestrant peptide (βARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOSl (SOS-Pro), and Raf (ΔN-Raf). Erk1/2 phosphorylation in response to α1B-AR stimulation is also attenuated by coexpression of N17- Ras, SOS-Pro, or ΔN-Raf, but not by coexpression of βARK1ct or by pretreatment with pertussis toxin. The α1B- and α2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the αlB-AR, and α2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant- negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogenactivated protein kinase phosphorylation after both αIB, and α2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium., and tyrosine kinase-dependent pathway for both G(i)- and G(q)/11/coupled receptors. These results indicate that in HEK-293 cells, the Gβγ subunit- mediated α3A-AR, and the Gα(q)/11-mediated α1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.

Authors
Della Rocca, GJ; Van Biesen, T; Daaka, Y; Luttrell, DK; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Della Rocca, GJ, Van Biesen, T, Daaka, Y, Luttrell, DK, Luttrell, LM, and Lefkowitz, RJ. "Ras-dependent mitogen-activated protein kinase activation by G protein- coupled receptors." Journal of Biological Chemistry 272.31 (August 1, 1997): 19125-19132.
Source
scopus
Published In
The Journal of biological chemistry
Volume
272
Issue
31
Publish Date
1997
Start Page
19125
End Page
19132
DOI
10.1074/jbc.272.31.19125

Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.

Authors
Della Rocca, GJ; van Biesen, T; Daaka, Y; Luttrell, DK; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Della Rocca, GJ, van Biesen, T, Daaka, Y, Luttrell, DK, Luttrell, LM, and Lefkowitz, RJ. "Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase." J Biol Chem 272.31 (August 1, 1997): 19125-19132.
PMID
9235901
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
31
Publish Date
1997
Start Page
19125
End Page
19132

Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity.

Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p < 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation approximately 2-fold (p < 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only approximately 25%, GRK2 inhibited ET-R signaling by 80% (p < 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.

Authors
Freedman, NJ; Ament, AS; Oppermann, M; Stoffel, RH; Exum, ST; Lefkowitz, RJ
MLA Citation
Freedman, NJ, Ament, AS, Oppermann, M, Stoffel, RH, Exum, ST, and Lefkowitz, RJ. "Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity." J Biol Chem 272.28 (July 11, 1997): 17734-17743.
PMID
9211925
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
28
Publish Date
1997
Start Page
17734
End Page
17743

Gene transfer strategies for augmenting cardiac function.

Recent transgenic as well as gene-targeted animal models have greatly increased our understanding of the molecular mechanisms of normal and compromised heart function. These studies have raised the possibility of using somatic gene transfer as a means for improving cardiac function. DNA transfer to a significant portion of the myocardium has thus far been difficult to accomplish. This review describes current efforts to achieve myocardial gene transfer in several model systems, with particular emphasis placed on adenovirus-mediated gene delivery, its possibilities, and current limitations. (Trend Cardiovasc Med 1997;7:145-150). © 1997, Elsevier Science Inc.

Authors
Peppel, K; Koch, WJ; Lefkowitz, RJ
MLA Citation
Peppel, K, Koch, WJ, and Lefkowitz, RJ. "Gene transfer strategies for augmenting cardiac function." Trends Cardiovasc Med 7.5 (July 1997): 145-150.
PMID
21235878
Source
pubmed
Published In
Trends in Cardiovascular Medicine
Volume
7
Issue
5
Publish Date
1997
Start Page
145
End Page
150
DOI
10.1016/S1050-1738(97)00028-5

Targeting G protein-coupled receptor kinases to their receptor substrates.

Authors
Stoffel, RH; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Stoffel, RH, Pitcher, JA, and Lefkowitz, RJ. "Targeting G protein-coupled receptor kinases to their receptor substrates." J Membr Biol 157.1 (May 1, 1997): 1-8. (Review)
PMID
9141353
Source
pubmed
Published In
The Journal of Membrane Biology
Volume
157
Issue
1
Publish Date
1997
Start Page
1
End Page
8

Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization.

Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the beta-adrenergic receptor kinase (betaARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the beta2-adrenergic receptor (beta2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that betaARK and arrestin proteins (beta-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the beta2AR to be phosphorylated by betaARK or to interact normally with beta-arrestin substantially reduces agonist-mediated sequestration. To evaluate betaARK and beta-arrestin regulation of beta2AR sequestration, we examined the relationship between betaARK and/or beta-arrestin expression and beta2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous beta-arrestin expression and beta2AR sequestration, whereas HEK 293 had the highest. Overexpression of beta-arrestin, but not betaARK, in COS cells increased the extent of wild-type beta2AR sequestration to levels observed in HEK 293 cells. However, a betaARK phosphorylation-impaired beta2AR mutant (Y326A) required the simultaneous overexpression of both betaARK and beta-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of betaARK and beta-arrestin expression. Moreover, an agonist-mediated translocation of wild-type beta2AR and endogenous beta-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular betaARK and arrestin proteins synergistically regulate beta2AR sequestration but also that beta-arrestins directly regulate beta2AR trafficking as well as desensitization.

Authors
Ménard, L; Ferguson, SS; Zhang, J; Lin, FT; Lefkowitz, RJ; Caron, MG; Barak, LS
MLA Citation
Ménard, L, Ferguson, SS, Zhang, J, Lin, FT, Lefkowitz, RJ, Caron, MG, and Barak, LS. "Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization." Mol Pharmacol 51.5 (May 1997): 800-808.
PMID
9145918
Source
pubmed
Published In
Molecular pharmacology
Volume
51
Issue
5
Publish Date
1997
Start Page
800
End Page
808

Potentiation of beta-adrenergic signaling by gene transfer.

The beta-adrenergic signaling cascade is an important regulator of myocardial function. Numerous abnormalities occur in this pathway and are associated with impaired cardiac contractility in patients with congestive heart failure (CHF). These signaling defects include downregulation of beta-adrenergic receptors (beta ARs) and increased levels of beta-adrenergic receptor kinase (beta ARK), an enzyme that phosphorylates and uncouples only agonist-bound receptors. Our laboratory has been testing the hypothesis that reversal of these beta-adrenergic defects may be able to restore cardiac inotropy to normal in patients with depressed systolic function. Transgenic mice with cardiac overexpression of beta 2ARs or an inhibitor of beta ARK have enhanced cardiac function as compared to wildtype littermates. Adenoviral vectors encoding the beta 2AR or beta ARK inhibitor potentiate beta AR signaling in cultured adult rabbit ventricular myocytes. However, a controversy has developed in the literature regarding whether increasing beta-adrenergic signaling would be beneficial or detrimental for patients with CHF. Those cautioning against this approach note that increased sympathetic activity is dangerous in CHF. Elevated catecholamine levels predict mortality and beta-agonists are not beneficial for survival, while recent studies suggest that beta-antagonists do improve outcome. Supporting these concerns is the demonstration that transgenic mice with cardiac overexpression of Gs alpha and enhanced myocardial responsiveness to isoproterenol develop myocardial fibrosis. This article summarizes this controversy; highlights important differences between overexpression of beta ARs or a beta ARK inhibitor, overexpression of Gs alpha, and administration of beta-agonists; and develops the hypothesis that these strategies may differ in their therapeutic efficacy in treating CHF.

Authors
Drazner, MH; Koch, WJ; Lefkowitz, RJ
MLA Citation
Drazner, MH, Koch, WJ, and Lefkowitz, RJ. "Potentiation of beta-adrenergic signaling by gene transfer." Proc Assoc Am Physicians 109.3 (May 1997): 220-227. (Review)
PMID
9154638
Source
pubmed
Published In
Proceedings of the Association of American Physicians
Volume
109
Issue
3
Publish Date
1997
Start Page
220
End Page
227

Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Authors
Daaka, Y; Pitcher, JA; Richardson, M; Stoffel, RH; Robishaw, JD; Lefkowitz, RJ
MLA Citation
Daaka, Y, Pitcher, JA, Richardson, M, Stoffel, RH, Robishaw, JD, and Lefkowitz, RJ. "Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases." Proc Natl Acad Sci U S A 94.6 (March 18, 1997): 2180-2185.
Website
http://hdl.handle.net/10161/7832
PMID
9122168
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
6
Publish Date
1997
Start Page
2180
End Page
2185

Functional analysis of myocardial performance in murine hearts overexpressing the human beta 2-adrenergic receptor.

Transgenic mice overexpressing the human beta 2-adrenergic receptor gene were compared with wild mice type in terms of cardiac function, using a modified work-performing isolated murine heart preparation and on-line computer analysis. A preload-dependent experiment was performed, in which venous return was gradually increased in 5 mmHg increments from 5 mmHg to 25 mmHg. At each preload, aortic flow, left atrial pressure and aortic pressure were measured in all hearts, and from these parameters stroke volume, contractility, and cardiac index (cardiac output divided by body weight in g) were calculated and compared between groups. At increasing preload levels, the heart rates ranged from 322 beats/min (+/-29) to 369 beats/min (+/-39) in control mice and from 469 beats/min (+/-36) to 540 beats/min (+/-39) in transgenic mice. Cardiac index increased from 138 microliters/min/g (+/-13) and 48 microliters/min/g (+/-5) for transgenic and control mice, respectively at 5 mmHg preload to 262 microliters/min/g (+/-51) and 167 microliters/min/g (+/-15), respectively at 20 mmHg preload. The contractility in the transgenic mice were significantly increased at lower preload levels compared to control mice (1420 mmHg/s +/- 204 v 1187 mmHg/s +/- 127). An increase in myocardial adrenergic receptor density (100-200 fold) leads to significantly higher indices of cardiac function in transgenic mice compared to control mice. The increased heart rate leading to a positive inotropic effect in the hearts of transgenic mice is, at least in part, due to the overexpression of adrenergic receptors. These findings suggest a possible alternative method of establishing a positive chronotropic and inotropic state without the use of pharmacological agents.

Authors
Bittner, HB; Chen, EP; Milano, CA; Lefkowitz, RJ; Van Trigt, P
MLA Citation
Bittner, HB, Chen, EP, Milano, CA, Lefkowitz, RJ, and Van Trigt, P. "Functional analysis of myocardial performance in murine hearts overexpressing the human beta 2-adrenergic receptor." J Mol Cell Cardiol 29.3 (March 1997): 961-967.
PMID
9152857
Source
pubmed
Published In
Journal of Molecular and Cellular Cardiology
Volume
29
Issue
3
Publish Date
1997
Start Page
961
End Page
967
DOI
10.1006/jmcc.1996.0339

Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation.

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and alpha2A adrenergic receptors or overexpression of Gbeta1gamma2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gbeta1gamma2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gbetagamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.

Authors
Luttrell, LM; Della Rocca, GJ; van Biesen, T; Luttrell, DK; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Della Rocca, GJ, van Biesen, T, Luttrell, DK, and Lefkowitz, RJ. "Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation." J Biol Chem 272.7 (February 14, 1997): 4637-4644.
PMID
9020193
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
7
Publish Date
1997
Start Page
4637
End Page
4644

Phosducin, potential role in modulation of olfactory signaling

Authors
Boekhoff, I; Touhara, K; Danner, S; Inglese, J; Lohse, MJ; Breer, H; Lefkowitz, RJ
MLA Citation
Boekhoff, I, Touhara, K, Danner, S, Inglese, J, Lohse, MJ, Breer, H, and Lefkowitz, RJ. "Phosducin, potential role in modulation of olfactory signaling." JOURNAL OF BIOLOGICAL CHEMISTRY 272.7 (February 14, 1997): 4606-4612.
PMID
9020189
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
272
Issue
7
Publish Date
1997
Start Page
4606
End Page
4612

Dual coupling of beta(2)-adrenergic receptor to G(i) as well as to G(s) in cardiac myocytes

Authors
Xiao, RP; Avdonin, P; Akliter, SA; Zhou, YY; Ziman, B; Cheng, H; Lefkowitz, RJ; Koch, WJ; Lakatta, EG
MLA Citation
Xiao, RP, Avdonin, P, Akliter, SA, Zhou, YY, Ziman, B, Cheng, H, Lefkowitz, RJ, Koch, WJ, and Lakatta, EG. "Dual coupling of beta(2)-adrenergic receptor to G(i) as well as to G(s) in cardiac myocytes." BIOPHYSICAL JOURNAL 72.2 (February 1997): TUAM2-TUAM2.
Source
wos-lite
Published In
Biophysical Journal
Volume
72
Issue
2
Publish Date
1997
Start Page
TUAM2
End Page
TUAM2

Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

Authors
Drazner, MH; Peppel, KC; Dyer, S; Grant, AO; Koch, WJ; Lefkowitz, RJ
MLA Citation
Drazner, MH, Peppel, KC, Dyer, S, Grant, AO, Koch, WJ, and Lefkowitz, RJ. "Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes." J Clin Invest 99.2 (January 15, 1997): 288-296.
Website
http://hdl.handle.net/10161/7831
PMID
9005997
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
99
Issue
2
Publish Date
1997
Start Page
288
End Page
296
DOI
10.1172/JCI119157

Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice.

Transgenic overexpression (40- to 100-fold) of the wild-type human beta2-adrenergic receptor in the hearts of mice leads to a marked increase in cardiac contractility, which is apparently due to the low level of spontaneous (i.e., agonist-independent) activity inherent in the receptor. Here we report that transgenic mice expressing a mutated constitutively active form of the receptor (CAM) show no such phenotype, owing to its modest expression (3-fold above endogenous cardiac beta-adrenergic receptor levels). Surprisingly, treatment of the animals with a variety of beta-adrenergic receptor ligands leads to a 50-fold increase in CAM beta2-adrenergic receptor expression, by stabilizing the CAM beta2-adrenergic receptor protein. Receptor up-regulation leads in turn to marked increases in adenylate cyclase activity, atrial tension determined in vitro, and indices of cardiac contractility determined in vivo. These results illustrate a novel mechanism for regulating physiological responses, i.e., ligand-induced stabilization of a constitutively active but inherently unstable protein.

Authors
Samama, P; Bond, RA; Rockman, HA; Milano, CA; Lefkowitz, RJ
MLA Citation
Samama, P, Bond, RA, Rockman, HA, Milano, CA, and Lefkowitz, RJ. "Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice." Proc Natl Acad Sci U S A 94.1 (January 7, 1997): 137-141.
Website
http://hdl.handle.net/10161/7830
PMID
8990174
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
1
Publish Date
1997
Start Page
137
End Page
141

The role of sequestration in G protein-coupled receptor resensitization. Regulation of beta2-adrenergic receptor dephosphorylation by vesicular acidification.

G protein-coupled receptor kinases phosphorylate the agonist occupied conformation of G protein-coupled receptors in the plasma membrane, leading to their desensitization. Receptor resensitization requires receptor dephosphorylation, a process which is mediated by a plasma and vesicular membrane-associated form of PP-2A. We present evidence that, like receptor phosphorylation, receptor dephosphorylation is tightly regulated, requiring a specific receptor conformation induced by vesicular acidification. In vitro, spontaneous dephosphorylation of phosphorylated receptors is observed only at acidic pH. Furthermore, in intact cells upon agonist stimulation, phosphorylated receptors traffic from the plasma membrane to vesicles where they become physically associated with the phosphatase and dephosphorylated. Treatment of cells with NH4Cl, which disrupts the acidic pH found in endosomal vesicles, blocks association of the receptors with the phosphatase and blocks receptor dephosphorylation. These findings suggest that a conformational change in the receptor induced by acidification of the endosomal vesicles is the key determinant regulating receptor dephosphorylation and resensitization.

Authors
Krueger, KM; Daaka, Y; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Krueger, KM, Daaka, Y, Pitcher, JA, and Lefkowitz, RJ. "The role of sequestration in G protein-coupled receptor resensitization. Regulation of beta2-adrenergic receptor dephosphorylation by vesicular acidification." J Biol Chem 272.1 (January 3, 1997): 5-8.
PMID
8995214
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
1
Publish Date
1997
Start Page
5
End Page
8

Transgenic manipulation of beta-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine

Authors
Korzick, DH; Xiao, RP; Ziman, BD; Koch, WJ; Lefkowitz, RJ; Lakatta, EG
MLA Citation
Korzick, DH, Xiao, RP, Ziman, BD, Koch, WJ, Lefkowitz, RJ, and Lakatta, EG. "Transgenic manipulation of beta-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine." AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY 272.1 (January 1997): H590-H596.
PMID
9038983
Source
wos-lite
Published In
American journal of physiology. Heart and circulatory physiology
Volume
272
Issue
1
Publish Date
1997
Start Page
H590
End Page
H596

Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization.

Dynamic regulation of G protein-coupled receptor signaling demands a coordinated balance between mechanisms leading to the generation, turning off and re-establishment of agonist-mediated signals. G protein-coupled receptor kinases (GRKs) and arrestin proteins not only mediate agonist-dependent G protein-coupled receptor desensitization, but also initiate the internalization (sequestration) of activated receptors, a process leading to receptor resensitization. Studies on the specificity of beta-arrestin functions reveal a multiplicity of G protein-coupled receptor endocytic pathways and suggest that beta-arrestins might serve as adaptors specifically targeting receptors for dynamin-dependent clathrin-mediated endocytosis. Moreover, inactivation of the GRK2 gene in mice has lead to the discovery of an unexpected role of GRK2 in cardiac development, further emphasizing the pleiotropic function of GRKs and arrestins.

Authors
Zhang, J; Ferguson, SS; Barak, LS; Aber, MJ; Giros, B; Lefkowitz, RJ; Caron, MG
MLA Citation
Zhang, J, Ferguson, SS, Barak, LS, Aber, MJ, Giros, B, Lefkowitz, RJ, and Caron, MG. "Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization." Receptors Channels 5.3-4 (1997): 193-199. (Review)
PMID
9606723
Source
pubmed
Published In
Receptors & channels
Volume
5
Issue
3-4
Publish Date
1997
Start Page
193
End Page
199

G protein-coupled receptor kinase 5 in cultured vascular smooth muscle cells and rat aorta: Regulation by angiotensin II and hypertension

GRK5, a recently cloned member of the G protein-coupled receptor kinase family, has been shown to phosphorylate and participate in the desensitization of angiotensin II (Ang II) type 1A (AT(1A)) receptors. In this study, the effect of angiotensin II on GRK5 expression was examined in cultured vascular smooth muscle cells and aortas of Ang II-infused hypertensive rats. In vascular smooth muscle cells, Ang II (100 nM) up- regulated GRK5 mRNA as early as 1 h, with a peak at 16 h. This up-regulation was dose- and calcium-dependent. The increase in GRK5 mRNA was reflected in a smaller increase in protein expression, which nonetheless had functional significance since AT1 receptor phosphorylation was increased and phospholipase C activation was decreased following prolonged incubation with Ang II. In aortas of Ang II-infused hypertensive rats, both GRK5 mRNA and protein levels increased ~3-fold compared with sham-operated rats at 5 and 7 days, respectively. This up-regulation was blocked either by losartan or by the nonspecific vasodilator hydralazine. Since a subpressor dose of Ang II did not increase GRK5 mRNA levels and norepinephrine infusion also increased GRK5 mRNA expression, we conclude that Ang II-induced GRK5 up-regulation in rat aortas may be due to hypertension per se. Hormone- and hemodynamic stress-induced GRK5 regulation may provide a novel molecular basis for long- term regulation of agonist sensitivity of vascular cells.

Authors
Ishizaka, N; Alexander, RW; Laursen, JB; Kai, H; Fukui, T; Oppermann, M; Lefkowitz, RJ; Lyons, PR; Griendling, KK
MLA Citation
Ishizaka, N, Alexander, RW, Laursen, JB, Kai, H, Fukui, T, Oppermann, M, Lefkowitz, RJ, Lyons, PR, and Griendling, KK. "G protein-coupled receptor kinase 5 in cultured vascular smooth muscle cells and rat aorta: Regulation by angiotensin II and hypertension." Journal of Biological Chemistry 272.51 (1997): 32482-32488.
PMID
9405459
Source
scival
Published In
The Journal of biological chemistry
Volume
272
Issue
51
Publish Date
1997
Start Page
32482
End Page
32488
DOI
10.1074/jbc.272.51.32482

Myocarondial Overexpression of Adrenergic Receptors and Receptor Kinases

Authors
Koch, WJ; Lefkowitz, RJ; Milano, CA; Akhter, SA; Rockmad, HA
MLA Citation
Koch, WJ, Lefkowitz, RJ, Milano, CA, Akhter, SA, and Rockmad, HA. "Myocarondial Overexpression of Adrenergic Receptors and Receptor Kinases." Advances in Pharmacology 42.C (1997): 502-506.
Source
scival
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Issue
C
Publish Date
1997
Start Page
502
End Page
506
DOI
10.1016/S1054-3589(08)60799-1

Molecular basis for gβγ-effector interaction: Structural correlation with ga binding site

This work is a collaborative study by several laboratories directed at localizing sites of interaction between Gβγ, an important mediator of transmembrane signaling, and numerous downstream partners. To this end we have targeted residues on Gβγ that contact the Get subunit in the heterotrimeric complex, since Ga often acts as a negative regulator of Gβy. A traditional alanine scanning mutagenesis approach was used to evaluate the role of each amino acid residue within Ga binding surface of Gβ in the ability of dimer to interact with G-protein receptor kinase 2 (βARK). inward rectifying potassium channel (GIRK), and the a subunits of Ca-channels The data presented supports the idea that Gβγ interacts uniquely with different effectors, although sites of interaction can overlap.

Authors
Skiba, N; Bae, H; Ford, C; Daaka, Y; Reuvenv, E; Shekter, L; Biesen, TV; Yang, C-S; Lambright, D; Sondek, J; Sigler, P; Miller, R; Jan, L; Lefkowitz, R; Hamm, H
MLA Citation
Skiba, N, Bae, H, Ford, C, Daaka, Y, Reuvenv, E, Shekter, L, Biesen, TV, Yang, C-S, Lambright, D, Sondek, J, Sigler, P, Miller, R, Jan, L, Lefkowitz, R, and Hamm, H. "Molecular basis for gβγ-effector interaction: Structural correlation with ga binding site." 1997.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1340

G protein-coupled receptors and their regulation

Seven membrane spanning domain G protein-coupled receptors represent the most ubiquitous and diverse group of membrane bound signaling receptors. Their function is regulated by two families of molecules, the G protein-coupled receptor kinases and the ß-arrestins which function in concert to desensitize receptor function after stimulation. The kinases phosphorylate only the activated forms of the receptor leading to the binding of ß-arrestin molecules which sterically interdict signal transduction to the G proteins. Some of the kinases such as the ß-adrenergic receptor kinase are largely cytosolic but are translocated to the membranes by interaction with membrane lipids such as PIP2 and membrane anchored proteins such as the Gß7 subunit complex. Different Gßy subunits associate with different members of the GRK family imparting a level of specificity to the interaction of these kinases with the receptors, ß-arrestin binding to the receptors also promotes their internalization into clathrin coated pits. Within internalized endosomes the receptors are dephosphorylated and resensitized by a special G protein-coupled receptor phosphatase, the activity of which toward the receptor is regulated by the uniquely low endosomal pH. Recent experiments also indicate that both ß-arrestin and the GRKs play previously unsuspected roles in cellular signaling cascades and these will be discussed.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "G protein-coupled receptors and their regulation." FASEB Journal 11.9 (1997): A873-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A873

Mechanisms of β-Adrenergic Receptor Desensitization and Resensitization

Authors
Lefkowitz, RJ; Pitcher, J; Krueger, K; Daaka, Y
MLA Citation
Lefkowitz, RJ, Pitcher, J, Krueger, K, and Daaka, Y. "Mechanisms of β-Adrenergic Receptor Desensitization and Resensitization." Advances in Pharmacology 42.C (1997): 416-420.
Source
scival
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
42
Issue
C
Publish Date
1997
Start Page
416
End Page
420
DOI
10.1016/S1054-3589(08)60777-2

Costimulation of adenylyl cyclase and phospholipase C by a mutant α(1B)-adrenergic receptor transgene promotes malignant transformation of thyroid follicular cells

Proliferation of thyroid follicular cells is controlled by three intracellular cascades [cAMP, inositol 1,4,5-triphosphate (IP 3)/Ca 2+/diacylglycerol (DAG), and tyrosine kinases] that are activated by distinct extracellular signals and receptors. We had previously generated a transgenic mouse model in which the cAMP cascade was permanently stimulated in thyroid cells by an adenosine A(2a) receptor (Tg-A(2a) R model). In the present work, we have generated a transgenic model characterized by the chronic stimulation of both adenylyl cyclase and phospholipase C in thyroid follicular cells. The bovine thyroglobulin gene promoter was used to direct the expression of a constitutively active mutant of the α(1B) adrenergic receptor, which is known to couple to both cascades in transfected cell lines. The expression of the transgene resulted, as expected, in the activation of phospholipase C and adenylyl cyclase, as demonstrated by the direct measurement of IP 3 and cAMP in thyroid tissue. The phenotype resulting from this dual stimulation included growth stimulation, hyperfunction, cell degeneracy attributed to the overproduction of free radicals, and the development of malignant nodules invading the capsule, muscles, and blood vessels. Differentiated metastases were found occasionally in old animals. The development of malignant lesions was more frequent and of earlier onset than in our previous Tg-A(2a)R model, in which only the cAMP cascade was stimulated. These observations demonstrate that the cAMP and IP 3/Ca 2+/DAG cascades can cooperate in vivo toward the development of thyroid follicular cell malignancies.

Authors
Ledent, C; Denef, J-F; Cottecchia, S; Lefkowitz, R; Dumont, J; Vassart, G; Parmentier, M
MLA Citation
Ledent, C, Denef, J-F, Cottecchia, S, Lefkowitz, R, Dumont, J, Vassart, G, and Parmentier, M. "Costimulation of adenylyl cyclase and phospholipase C by a mutant α(1B)-adrenergic receptor transgene promotes malignant transformation of thyroid follicular cells." Endocrinology 138.1 (1997): 369-378.
Website
http://hdl.handle.net/10161/7829
PMID
8977426
Source
scival
Published In
Endocrinology
Volume
138
Issue
1
Publish Date
1997
Start Page
369
End Page
378
DOI
10.1210/en.138.1.369

Transgenic manipulation of β-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine

To determine the direct functional significance of the β-adrenergic receptor (AR) kinase 1 (βARK1) on myocardial performance in the absence of tonic sympathoadrenal neural activation and mechanical loading, we measured the contractile responses to acute β1-AR stimulation in left ventricular myocytes isolated from nontransgenic control (NTG) and transgenic mice overexpressing either βARK1 (TGβK12) or a βARK1 inhibitor (TGMini27). Contractile response to five concentrations (10-8-10-7 M) of the β1-AR agonist norepinephrine (NE) plus prazosin (10-6 M) was measured after a 60- s rest, i.e., rested-state contraction (RSC), and during steady-state contraction (SSC) stimulation at 0.5 Hz (23°C). At baseline, resting cell length was significantly greater in TGβK12 myocytes (P < 0.05); however, there were no significant differences in RSC or SSC among NTG, TGβK12, or TGMini27 mice. On the other hand, both the dose-response curve and kinetics for the NE-induced SSC response normalized to RSC (SSC/RSC) were significantly different among experimental groups (P < 0.001). Specifically, maximal SSC induced by NE in myocytes isolated from TGβK12 was only 70% of the response observed in NTG cells and 50% of the response measured in TGMini27. These data suggest that 1) in the absence of circulating catecholamines or basal sympathetic tone, βARK1 actions in single myocytes are minimal, and 2) substantial functional βARK1 modulation of β1AR signaling occurs in cardiac myocytes even during short-term β1-AR stimulation. These results are consistent with a role for agonist-induced phosphorylation and desensitization of cardiac β1-ARs by βARK1 in single myocytes and highlight the potential functional importance of βARK1 as a critical determinant of the cardiac β1-AR contractile response.

Authors
Korzick, DH; Xiao, R-P; Ziman, BD; Koch, WJ; Lefkowitz, RJ; Lakatta, EG
MLA Citation
Korzick, DH, Xiao, R-P, Ziman, BD, Koch, WJ, Lefkowitz, RJ, and Lakatta, EG. "Transgenic manipulation of β-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine." American Journal of Physiology - Heart and Circulatory Physiology 272.1 41-1 (1997): H590-H596.
Source
scival
Published In
American journal of physiology. Heart and circulatory physiology
Volume
272
Issue
1 41-1
Publish Date
1997
Start Page
H590
End Page
H596

Cardiac function in genetically engineered mice with altered adrenergic receptor signaling

In disease states such as heart failure, catecholamines released from sympathetic nerve endings and the adrenal medulla play a central role in the adaptive and maladaptive physiological response to altered tissue perfusion. G protein-coupled receptors are importantly involved in myocardial growth and the regulation of contractility. The adrenergic receptors themselves are regulated by a set of specific kinases, termed the G protein-coupled receptor kinases. The study of complex systems in vivo has recently been advanced by the development of transgenic and gene-targeted 'knockout' mouse models. Combining transgenic technology with sophisticated physiological measurements of cardiac function is an extremely powerful strategy for studying the regulation of myocardial contractility in normal animals and in models of disease states. The purpose of this review is to summarize current knowledge about the regulation of cardiovascular homeostasis involving signaling pathways through stimulation of adrenergic receptors.

Authors
Rockman, HA; Koch, WJ; Lefkowitz, RJ
MLA Citation
Rockman, HA, Koch, WJ, and Lefkowitz, RJ. "Cardiac function in genetically engineered mice with altered adrenergic receptor signaling." American Journal of Physiology - Heart and Circulatory Physiology 272.4 41-4 (1997): H1553-H1559.
PMID
9139936
Source
scival
Published In
American journal of physiology. Heart and circulatory physiology
Volume
272
Issue
4 41-4
Publish Date
1997
Start Page
H1553
End Page
H1559

Gβγ subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin- sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of G(i)-coupled lysophosphatidic acid and α2A adrenergic receptors or overexpression of Gβ1γ2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein hand contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following G(i)-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gβ1γ2 subunit-mediated and G(i)-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The G(i)-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophostidic and stimulates binding of EGF receptor to a GST fushion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gβγ subunit mediated activation of Src family nonreceptor tyrosine kinases can account for the G(i))-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.

Authors
Luttrell, LM; Rocca, GJD; Biesen, TV; Luttrell, DK; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Rocca, GJD, Biesen, TV, Luttrell, DK, and Lefkowitz, RJ. "Gβγ subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation." Journal of Biological Chemistry 272.7 (1997): 4637-4644.
Source
scival
Published In
Journal of Biological Chemistry
Volume
272
Issue
7
Publish Date
1997
Start Page
4637
End Page
4644
DOI
10.1074/jbc.272.7.4637

G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors.

G-protein-coupled receptors that mediate cellular responses to a variety of humoral, endothelial-, or platelet-derived substances are able to stimulate MAP kinase activity. In transfected model systems, G-protein-coupled receptors that couple to pertussis toxin-insensitive G proteins of the Gq/11 family mediate this activation predominantly via a PKC-dependent mechanism. In contrast, activation of MAP kinase by receptors that couple to pertussis toxin-sensitive Gi proteins is PKC-independent and requires downstream activation of the low-molecular-weight G protein, Ras. This pathway can be inhibited by coexpression of peptides that sequester Gbetagamma subunits, and is mimicked by overexpression of Gbetagamma subunits. This Ras-dependent MAP kinase activation requires tyrosine phosphorylation of "docking proteins," including the shc adapter protein, and depends upon recruitment of Grb2/Sos1 complexes to the plasma membrane, thus resembling the pathway of MAP kinase activation employed by the receptor tyrosine kinases. Other molecules, including PI-3-kinases and phosphotyrosine phosphatases, probably also contribute to Gbetagamma-subunit-mediated assembly of a mitogenic signaling complex. Identification of the G-protein-coupled, receptor-regulated tyrosine kinase(s), and the means by which the mitogenic signaling complex is assembled at the plasma membrane, remain subjects of further study.

Authors
Luttrell, LM; van Biesen, T; Hawes, BE; Koch, WJ; Krueger, KM; Touhara, K; Lefkowitz, RJ
MLA Citation
Luttrell, LM, van Biesen, T, Hawes, BE, Koch, WJ, Krueger, KM, Touhara, K, and Lefkowitz, RJ. "G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors." Adv Second Messenger Phosphoprotein Res 31 (1997): 263-277. (Review)
PMID
9344257
Source
pubmed
Published In
Advances in Second Messenger and Phosphoprotein Research
Volume
31
Publish Date
1997
Start Page
263
End Page
277

Mini-Review - Molecular mechanisms of G protein-coupled receptor signaling: Role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization

Authors
Zhang, J; Ferguson, SSG; Barak, LS; Aber, MJ; Giros, B; Lefkowitz, RJ; Caron, MG
MLA Citation
Zhang, J, Ferguson, SSG, Barak, LS, Aber, MJ, Giros, B, Lefkowitz, RJ, and Caron, MG. "Mini-Review - Molecular mechanisms of G protein-coupled receptor signaling: Role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization." 1997.
Source
wos-lite
Published In
Receptors & channels
Volume
5
Issue
3-4
Publish Date
1997
Start Page
193
End Page
199

Cloning, characterization, and chromosomal localization of rec1.3, a member of the G-protein-coupled receptor family highly expressed in brain.

During a project to identify G-protein-coupled receptors (GPCR) expressed within taste buds, we have isolated a novel receptor-like sequence. The full length sequence of this receptor (rec1.3) has been obtained in both cow and mouse. Rec1.3 bears little sequence similarity to any GPCR whose ligand is known: the closest identity (33%) is to the orphan receptor edg-1. In cow, rec1.3 is expressed most prominently in the brain, with moderate expression in testis and tongue; in the mouse the expression is more widespread. No specific binding for a range of ligands was detected when the mouse coding sequence was expressed in eukaryotic cells. In situ hybridization showed that rec1.3 is widely expressed throughout the mouse brain and is highly expressed in localized regions of the hindbrain, midbrain and hypothalamus. The rec1.3 gene was localized to the centromeric region of chromosome 4 in mouse, a region associated with neonatal seizures.

Authors
Macrae, AD; Premont, RT; Jaber, M; Peterson, AS; Lefkowitz, RJ
MLA Citation
Macrae, AD, Premont, RT, Jaber, M, Peterson, AS, and Lefkowitz, RJ. "Cloning, characterization, and chromosomal localization of rec1.3, a member of the G-protein-coupled receptor family highly expressed in brain." Brain Res Mol Brain Res 42.2 (December 1996): 245-254.
PMID
9013780
Source
pubmed
Published In
Molecular Brain Research
Volume
42
Issue
2
Publish Date
1996
Start Page
245
End Page
254

Mitogenic signaling via G protein-coupled receptors.

Authors
van Biesen, T; Luttrell, LM; Hawes, BE; Lefkowitz, RJ
MLA Citation
van Biesen, T, Luttrell, LM, Hawes, BE, and Lefkowitz, RJ. "Mitogenic signaling via G protein-coupled receptors." Endocr Rev 17.6 (December 1996): 698-714. (Review)
Website
http://hdl.handle.net/10161/5921
PMID
8969974
Source
pubmed
Published In
Endocrine reviews
Volume
17
Issue
6
Publish Date
1996
Start Page
698
End Page
714
DOI
10.1210/edrv-17-6-698

Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

Authors
Jaber, M; Koch, WJ; Rockman, H; Smith, B; Bond, RA; Sulik, KK; Ross, J; Lefkowitz, RJ; Caron, MG; Giros, B
MLA Citation
Jaber, M, Koch, WJ, Rockman, H, Smith, B, Bond, RA, Sulik, KK, Ross, J, Lefkowitz, RJ, Caron, MG, and Giros, B. "Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function." Proc Natl Acad Sci U S A 93.23 (November 12, 1996): 12974-12979.
Website
http://hdl.handle.net/10161/7833
PMID
8917529
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
23
Publish Date
1996
Start Page
12974
End Page
12979

Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma-subunit-initiated pathway.

Serotonin (5-HT) is a potent mitogen in many cells types, an action which is frequently mediated through pertussis toxin-sensitive G proteins. In the current study, we used pharmacological inhibitors and dominant negative signaling constructs to delineate elements which participate in the activation of MAPK, a growth-associated mitogen-activated protein kinase, by human G protein-coupled 5-HT1A receptor transfected into CHO-K1 cells in a stable manner. The activation pathway does not directly involve phorbol ester-sensitive protein kinase C types, but does require (i) pertussis toxin-sensitive G protein beta gamma-subunits, (ii) a staurosporine- and genistein-sensitive protein kinase, (iii) phosphoinositide-3'-kinase activity, (iv) activation of Sos in a multimolecular complex that contains p46Shc, and p52Shc, and Grb2, (v) the GTPase p21Ras, and (vi) the protein kinase p74Raf-1. These data demonstrate that the 5-HT1A receptor mediates MAPK activity by convergence upon a common activation pathway that is shared with receptor tyrosine kinases.

Authors
Garnovskaya, MN; van Biesen, T; Hawe, B; Casañas Ramos, S; Lefkowitz, RJ; Raymond, JR
MLA Citation
Garnovskaya, MN, van Biesen, T, Hawe, B, Casañas Ramos, S, Lefkowitz, RJ, and Raymond, JR. "Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma-subunit-initiated pathway." Biochemistry 35.43 (October 29, 1996): 13716-13722.
PMID
8901512
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
43
Publish Date
1996
Start Page
13716
End Page
13722
DOI
10.1021/bi961764n

Functional effects of myocardial alpha-adrenergic receptor overexpression in transgenic mice.

Authors
Milano, CA; Akhter, SA; Koch, WJ; Dolber, PC; Rindt, H; Grupp, IL; Lefkowitz, RJ
MLA Citation
Milano, CA, Akhter, SA, Koch, WJ, Dolber, PC, Rindt, H, Grupp, IL, and Lefkowitz, RJ. "Functional effects of myocardial alpha-adrenergic receptor overexpression in transgenic mice." October 15, 1996.
Source
wos-lite
Published In
Circulation
Volume
94
Issue
8
Publish Date
1996
Start Page
2374
End Page
2374

Desensitization of adenosine A1 receptors with cardiac overexpression of the G protein-coupled receptor kinase 5 in transgenic mice.

Authors
Tsimikas, S; Koch, WJ; Lefkowitz, RJ; Rockman, HA
MLA Citation
Tsimikas, S, Koch, WJ, Lefkowitz, RJ, and Rockman, HA. "Desensitization of adenosine A1 receptors with cardiac overexpression of the G protein-coupled receptor kinase 5 in transgenic mice." CIRCULATION 94.8 (October 15, 1996): 2375-2375.
Source
wos-lite
Published In
Circulation
Volume
94
Issue
8
Publish Date
1996
Start Page
2375
End Page
2375

Blunted contractility in conscious transgenic mice overexpressing the G protein coupled receptor kinase-5

Authors
Rockman, HA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Rockman, HA, Lefkowitz, RJ, and Koch, WJ. "Blunted contractility in conscious transgenic mice overexpressing the G protein coupled receptor kinase-5." CIRCULATION 94.8 (October 15, 1996): 2251-2251.
Source
wos-lite
Published In
Circulation
Volume
94
Issue
8
Publish Date
1996
Start Page
2251
End Page
2251

Genetic manipulation of the beta-adrenergic receptor kinase (beta ARK1) modifies contractile response to beta(1)-adrenergic stimulation in single cardiac myocytes

Authors
Korzick, DH; Xiao, RP; Ziman, BD; Koch, MJ; Lefkowitz, RJ; Lakatta, EG
MLA Citation
Korzick, DH, Xiao, RP, Ziman, BD, Koch, MJ, Lefkowitz, RJ, and Lakatta, EG. "Genetic manipulation of the beta-adrenergic receptor kinase (beta ARK1) modifies contractile response to beta(1)-adrenergic stimulation in single cardiac myocytes." CIRCULATION 94.8 (October 15, 1996): 1676-1676.
Source
wos-lite
Published In
Circulation
Volume
94
Issue
8
Publish Date
1996
Start Page
1676
End Page
1676

Specific interactions between adrenergic receptors and receptor kinases in the hearts of hybrid transgenic mice

Authors
Akhter, SA; Milano, CA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Akhter, SA, Milano, CA, Lefkowitz, RJ, and Koch, WJ. "Specific interactions between adrenergic receptors and receptor kinases in the hearts of hybrid transgenic mice." October 15, 1996.
Source
wos-lite
Published In
Circulation
Volume
94
Issue
8
Publish Date
1996
Start Page
2373
End Page
2373

Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies.

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 11707-11710). Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.

Authors
Pitcher, JA; Fredericks, ZL; Stone, WC; Premont, RT; Stoffel, RH; Koch, WJ; Lefkowitz, RJ
MLA Citation
Pitcher, JA, Fredericks, ZL, Stone, WC, Premont, RT, Stoffel, RH, Koch, WJ, and Lefkowitz, RJ. "Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies." J Biol Chem 271.40 (October 4, 1996): 24907-24913.
PMID
8798768
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
40
Publish Date
1996
Start Page
24907
End Page
24913

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Authors
Rockman, HA; Choi, DJ; Rahman, NU; Akhter, SA; Lefkowitz, RJ; Koch, WJ
MLA Citation
Rockman, HA, Choi, DJ, Rahman, NU, Akhter, SA, Lefkowitz, RJ, and Koch, WJ. "Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice." Proc Natl Acad Sci U S A 93.18 (September 3, 1996): 9954-9959.
Website
http://hdl.handle.net/10161/7834
PMID
8790438
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
18
Publish Date
1996
Start Page
9954
End Page
9959

Myocardial beta-adrenergic receptor signaling in vivo: insights from transgenic mice.

Heart failure is a problem of increasing importance in cardiovascular medicine. An important characteristic of heart failure is reduced agonist-stimulated adenylyl cyclase activity (receptor desensitization) due to both diminished receptor number (receptor downregulation) and impaired receptor function (receptor uncoupling). These changes in the section-adrenergic receptor (section-AR) system may in part account for some of the abnormalities of contractile function in this disease. Myocardial contraction is closely regulated by G protein coupled beta-adrenergic receptors through the action of the second messenger cAMP. The beta-adrenergic receptors themselves are regulated by a set of specific kinases, termed the G-protein-coupled receptor kinases. The study of this complex system in vivo has recently been advanced by the development of transgenic and gene targeted ("knockout") mouse models. Combining transgenic technology with sophisticated physiological measurements of cardiac hemodynamics is an extremely powerful strategy to study the regulation of myocardial contractility in the normal and failing heart.

Authors
Rockman, HA; Koch, WJ; Milano, CA; Lefkowitz, RJ
MLA Citation
Rockman, HA, Koch, WJ, Milano, CA, and Lefkowitz, RJ. "Myocardial beta-adrenergic receptor signaling in vivo: insights from transgenic mice." J Mol Med (Berl) 74.9 (September 1996): 489-495. (Review)
PMID
8892053
Source
pubmed
Published In
Journal of Molecular Medicine
Volume
74
Issue
9
Publish Date
1996
Start Page
489
End Page
495

Specific alpha-subunits of the G(i/o/z) family of hererotrimeric G-proteins mediate acute agonist-induced activation of fibroblast NHE.

Authors
Garnovskaya, MN; vanBiesen, T; Lefkowitz, RJ; Raymond, JR
MLA Citation
Garnovskaya, MN, vanBiesen, T, Lefkowitz, RJ, and Raymond, JR. "Specific alpha-subunits of the G(i/o/z) family of hererotrimeric G-proteins mediate acute agonist-induced activation of fibroblast NHE." JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 7.9 (September 1996): A2144-A2144.
Source
wos-lite
Published In
Journal of the American Society of Nephrology : JASN
Volume
7
Issue
9
Publish Date
1996
Start Page
A2144
End Page
A2144

Divergent pertussis toxin-sensitive pathways for 5-HT1A receptor activation of NHE & ERK

Authors
Garnovskaya, MN; vanBiesen, T; Hawes, B; Lefkowitz, RJ; Raymond, JR
MLA Citation
Garnovskaya, MN, vanBiesen, T, Hawes, B, Lefkowitz, RJ, and Raymond, JR. "Divergent pertussis toxin-sensitive pathways for 5-HT1A receptor activation of NHE & ERK." JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 7.9 (September 1996): A2143-A2143.
Source
wos-lite
Published In
Journal of the American Society of Nephrology : JASN
Volume
7
Issue
9
Publish Date
1996
Start Page
A2143
End Page
A2143

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases.

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.

Authors
Luttrell, LM; Hawes, BE; van Biesen, T; Luttrell, DK; Lansing, TJ; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Hawes, BE, van Biesen, T, Luttrell, DK, Lansing, TJ, and Lefkowitz, RJ. "Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases." J Biol Chem 271.32 (August 9, 1996): 19443-19450.
PMID
8702633
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
32
Publish Date
1996
Start Page
19443
End Page
19450

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Authors
Oppermann, M; Diversé-Pierluissi, M; Drazner, MH; Dyer, SL; Freedman, NJ; Peppel, KC; Lefkowitz, RJ
MLA Citation
Oppermann, M, Diversé-Pierluissi, M, Drazner, MH, Dyer, SL, Freedman, NJ, Peppel, KC, and Lefkowitz, RJ. "Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases." Proc Natl Acad Sci U S A 93.15 (July 23, 1996): 7649-7654.
Website
http://hdl.handle.net/10161/7835
PMID
8755530
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
15
Publish Date
1996
Start Page
7649
End Page
7654

Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor.

Rapid desensitization of G protein-coupled receptors is mediated, at least in part, by their phosphorylation by the G protein-coupled receptor kinases (GRKs). However, only in the case of rhodopsin have the actual sites of receptor phosphorylation been unambiguously determined. Although previous studies have implicated the cytoplasmic tail of the beta2-adrenergic receptor (beta2AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown. Here we report the identification of the sites of GRK2- and GRK5-mediated beta2AR phosphorylation. The phosphorylation sites of both serine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the beta2AR. Of the seven phosphorylatable residues within this peptide, six are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (approximately 1.0 mol Pi/mol receptor). In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate between sites are observed for GRK2 and GRK5. Increasing the stoichiometry of GRK2-mediated beta2AR phosphorylation from approximately 1.0 to 5.0 mol Pi/mol receptor increases the stoichiometry of phosphorylation of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the beta2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.

Authors
Fredericks, ZL; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Fredericks, ZL, Pitcher, JA, and Lefkowitz, RJ. "Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor." J Biol Chem 271.23 (June 7, 1996): 13796-13803.
PMID
8662852
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
23
Publish Date
1996
Start Page
13796
End Page
13803

Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C.

The type 1A angiotensin II receptor (AT1A-R), which mediates cardiovascular effects of angiotensin II, has been shown to undergo rapid agonist-induced desensitization. We investigated the potential role of second messenger-activated kinases and G protein-coupled receptor kinases (GRKs) in the regulation of this receptor. In 293 cells transfected with the AT1A-R, a 3-min challenge with angiotensin II engendered a 46% decrease in subsequent angiotensin II-stimulated phosphoinositide hydrolysis in intact cells. This agonist-induced desensitization correlated temporally and dose-dependently with the phosphorylation of the receptor to a stoichiometry of 1 mol of phosphate/mol of receptor, as assessed by immunoprecipitation of receptors from cells metabolically labeled with 32Pi. Agonist-induced receptor phosphorylation was reduced by 40-50% by either overexpression of a dominant negative K220R mutant GRK2 or treatment of the cells with the protein kinase C (PKC) inhibitor staurosporine, in a virtually additive fashion. Cellular overexpression of GRK2K220R not only inhibited agonist-induced AT1A-R phosphorylation, but also prevented receptor desensitization, as assessed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition by staurosporine did not affect homologous desensitization of the AT1A-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the agonist-induced AT1A-R phosphorylation 1.5- to 1.7-fold (p < 0.001). These findings suggest a role for receptor phosphorylation by one or several GRKs in the rapid agonist-induced desensitization of the AT1A-R.

Authors
Oppermann, M; Freedman, NJ; Alexander, RW; Lefkowitz, RJ
MLA Citation
Oppermann, M, Freedman, NJ, Alexander, RW, and Lefkowitz, RJ. "Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C." J Biol Chem 271.22 (May 31, 1996): 13266-13272.
PMID
8662816
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
22
Publish Date
1996
Start Page
13266
End Page
13272

Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway.

The beta gamma-subunit of Gi mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which G beta gamma mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in G beta gamma-mediated MAP kinase activation. We show that Gi-coupled receptor- and G beta gamma-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by over expression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit Gi-coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by over-expression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the G beta gamma-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation.

Authors
Hawes, BE; Luttrell, LM; van Biesen, T; Lefkowitz, RJ
MLA Citation
Hawes, BE, Luttrell, LM, van Biesen, T, and Lefkowitz, RJ. "Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway." J Biol Chem 271.21 (May 24, 1996): 12133-12136.
PMID
8647803
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
21
Publish Date
1996
Start Page
12133
End Page
12136

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

Authors
Ménard, L; Ferguson, SS; Barak, LS; Bertrand, L; Premont, RT; Colapietro, AM; Lefkowitz, RJ; Caron, MG
MLA Citation
Ménard, L, Ferguson, SS, Barak, LS, Bertrand, L, Premont, RT, Colapietro, AM, Lefkowitz, RJ, and Caron, MG. "Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration." Biochemistry 35.13 (April 2, 1996): 4155-4160.
PMID
8672451
Source
pubmed
Published In
Biochemistry
Volume
35
Issue
13
Publish Date
1996
Start Page
4155
End Page
4160
DOI
10.1021/bi952961+

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

Authors
Rockman, HA; Hamilton, RA; Jones, LR; Milano, CA; Mao, L; Lefkowitz, RJ
MLA Citation
Rockman, HA, Hamilton, RA, Jones, LR, Milano, CA, Mao, L, and Lefkowitz, RJ. "Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein." J Clin Invest 97.7 (April 1, 1996): 1618-1623.
Website
http://hdl.handle.net/10161/5929
PMID
8601626
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
97
Issue
7
Publish Date
1996
Start Page
1618
End Page
1623
DOI
10.1172/JCI118587

Transgenic manipulation of myocardial G protein-coupled receptors and receptor kinases.

Authors
Koch, WJ; Milano, CA; Lefkowitz, RJ
MLA Citation
Koch, WJ, Milano, CA, and Lefkowitz, RJ. "Transgenic manipulation of myocardial G protein-coupled receptors and receptor kinases." Circ Res 78.4 (April 1996): 511-516. (Review)
PMID
8635207
Source
pubmed
Published In
Circulation Research
Volume
78
Issue
4
Publish Date
1996
Start Page
511
End Page
516

Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants.

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

Authors
Premont, RT; Macrae, AD; Stoffel, RH; Chung, N; Pitcher, JA; Ambrose, C; Inglese, J; MacDonald, ME; Lefkowitz, RJ
MLA Citation
Premont, RT, Macrae, AD, Stoffel, RH, Chung, N, Pitcher, JA, Ambrose, C, Inglese, J, MacDonald, ME, and Lefkowitz, RJ. "Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants." J Biol Chem 271.11 (March 15, 1996): 6403-6410.
PMID
8626439
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
11
Publish Date
1996
Start Page
6403
End Page
6410

G protein-coupled receptors and receptor kinases: from molecular biology to potential therapeutic applications.

Authors
Lefkowitz, RJ
MLA Citation
Lefkowitz, RJ. "G protein-coupled receptors and receptor kinases: from molecular biology to potential therapeutic applications." Nat Biotechnol 14.3 (March 1996): 283-286. (Review)
PMID
9630887
Source
pubmed
Published In
Nature Biotechnology
Volume
14
Issue
3
Publish Date
1996
Start Page
283
End Page
286
DOI
10.1038/nbt0396-283

An essential role for SRC tyrosine kinase SHC adapter protein complexes in G beta gamma subunit-mediated mitogen-activated protein kinase activation.

Authors
Luttrell, LM; Hawes, BE; vanBiesen, T; Luttrell, DK; Lefkowitz, RJ
MLA Citation
Luttrell, LM, Hawes, BE, vanBiesen, T, Luttrell, DK, and Lefkowitz, RJ. "An essential role for SRC tyrosine kinase SHC adapter protein complexes in G beta gamma subunit-mediated mitogen-activated protein kinase activation." JOURNAL OF INVESTIGATIVE MEDICINE 44.3 (March 1996): A271-A271.
Source
wos-lite
Published In
Journal of Investigative Medicine
Volume
44
Issue
3
Publish Date
1996
Start Page
A271
End Page
A271

Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases.

The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.

Authors
Tiberi, M; Nash, SR; Bertrand, L; Lefkowitz, RJ; Caron, MG
MLA Citation
Tiberi, M, Nash, SR, Bertrand, L, Lefkowitz, RJ, and Caron, MG. "Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases." J Biol Chem 271.7 (February 16, 1996): 3771-3778.
PMID
8631993
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
7
Publish Date
1996
Start Page
3771
End Page
3778

G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism.

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyzed G(o) alpha-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway.

Authors
van Biesen, T; Hawes, BE; Raymond, JR; Luttrell, LM; Koch, WJ; Lefkowitz, RJ
MLA Citation
van Biesen, T, Hawes, BE, Raymond, JR, Luttrell, LM, Koch, WJ, and Lefkowitz, RJ. "G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism." J Biol Chem 271.3 (January 19, 1996): 1266-1269.
PMID
8576109
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
3
Publish Date
1996
Start Page
1266
End Page
1269

Production of adrenergic receptors in yeast.

Using a recombinant yeast strain expressing human beta 2 adrenergic receptors under a galactose-inducible promoter, we established conditions for receptor production in 1-15 liter fermenter culture. Crucial factors contributing to consistent high-level expression included the use of selective glucose-free medium, the maintenance of the pH of the culture at 7.2-7.5 and the presence of an antagonist. The expression strategy and production conditions used with the beta 2 adrenergic receptor were then employed to express the human alpha 2-C2 adrenergic receptor in Saccharomyces cerevisiae. Galactose-induced yeast cells displayed specific, high-affinity [3H]rauwolscine binding and contained a 50-kDa species recognized by an alpha 2-C2 receptor specific antiserum. In fermenter culture, up to 10(5) high-affinity [3H]rauwolscine binding sites per cell (corresponding to 30-60 pmol/mg of protein) were obtained. The high expression level combined with relative ease and low cost of scaling-up make yeast a promising alternative to mammalian cells for the production of adrenergic and other G-protein-coupled receptors for structural studies.

Authors
Sizmann, D; Kuusinen, H; Keränen, S; Lomasney, J; Caron, MG; Lefkowitz, RJ; Keinänen, K
MLA Citation
Sizmann, D, Kuusinen, H, Keränen, S, Lomasney, J, Caron, MG, Lefkowitz, RJ, and Keinänen, K. "Production of adrenergic receptors in yeast." Receptors Channels 4.3 (1996): 197-203.
PMID
9014242
Source
pubmed
Published In
Receptors & channels
Volume
4
Issue
3
Publish Date
1996
Start Page
197
End Page
203

Desensitization of G protein-coupled receptors.

Waning responsiveness to continuous or repeated stimulation constitutes the phenomenon of desensitization, which pervades biological systems. Over the last several years, molecular mechanisms for desensitization of cellular signaling through G protein-coupled receptors have been delineated, particularly at the level of the receptors themselves. This review focuses on those aspects of G protein-coupled receptor desensitization which occur within minutes of agonist exposure. Agonist-dependent desensitization of these receptors can reduce their signaling responsiveness to maximum stimulation by up to 70-80%; indeed, in some receptor systems, the process of receptor desensitization appears to effect the termination of the cellular signaling response. Agonist-induced desensitization involves phosphorylation of G protein-coupled receptors by two currently recognized classes of serine/threonine protein kinases. Second messenger-dependent kinases, phosphorylating a variety of proteins, mediate a generalized cellular hyporesponsiveness termed heterologous desensitization. G protein-coupled receptor kinases (GRKs) phosphorylate specifically agonist-occupied, or activated, receptors, and thereby initiate receptor-specific, or homologous, desensitization. GRK-mediated receptor phosphorylation facilitates the binding of an inhibitory arrestin protein to the phosphorylated receptor, an event which substantially impairs receptor signaling. The GRK family comprises six, and the arrestin family comprises four known members. Each GRK demonstrates the ability to phosphorylate a limited number of model receptor substrates, but the widespread expression of several GRKs and the two somatic arrestins suggest that GRK-initiated homologous desensitization is of general importance to a wide range of G protein-coupled receptors. Exploration of the substrate specificity for GRKs and the ligand specificity for arrestins remains in its early stages. Currently, GRKs can most readily be differentiated by the mechanisms each employs to associate with the plasma membrane. Accumulating data from human disease states and transgenic mice attest to the physiologic significance of GRK-initiated receptor desensitization.

Authors
Freedman, NJ; Lefkowitz, RJ
MLA Citation
Freedman, NJ, and Lefkowitz, RJ. "Desensitization of G protein-coupled receptors." Recent Prog Horm Res 51 (1996): 319-351. (Review)
PMID
8701085
Source
pubmed
Published In
Recent progress in hormone research
Volume
51
Publish Date
1996
Start Page
319
End Page
351

The third beta is not the charm.

Authors
Bond, RA; Lefkowitz, RJ
MLA Citation
Bond, RA, and Lefkowitz, RJ. "The third beta is not the charm." The Journal of clinical investigation 98.2 (1996): 241--.
Website
http://hdl.handle.net/10161/7836
PMID
8755628
Source
scival
Published In
Journal of Clinical Investigation
Volume
98
Issue
2
Publish Date
1996
Start Page
241-
DOI
10.1172/JCI118783

G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced Ca2+ channel inhibition

G protein-coupled receptors are essential signaling molecules at sites of synaptic transmission. Here, we explore the mechanisms responsible for the use-dependent termination of metabotropic receptor signaling in embryonic sensory neurons. We report that the inhibition of voltage-dependent Ca2+ channels mediated by α2-adrenergic receptors desensitizes slowly with prolonged exposure to the transmitter and that the desensitization is mediated by a G protein-coupled receptor kinase (GRK). Intracellular introduction of recombinant, purified kinases or synthetic blocking peptides into individual neurons demonstratesthe specific involvement of a GRK3-like protein. These results suggest that GRK-mediated termination of receptor-G protein coupling is likely to regulate synaptic strength and, as such, may provide one effective mechanism for depression of synaptic transmission.

Authors
Diversé-Pierluissi, M; Inglese, J; Stoffel, RH; Lefkowitz, RJ; Dunlap, K
MLA Citation
Diversé-Pierluissi, M, Inglese, J, Stoffel, RH, Lefkowitz, RJ, and Dunlap, K. "G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced Ca2+ channel inhibition." Neuron 16.3 (1996): 579-585.
PMID
8785055
Source
scival
Published In
Neuron
Volume
16
Issue
3
Publish Date
1996
Start Page
579
End Page
585
DOI
10.1016/S0896-6273(00)80077-X

Go-protein α-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the α2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein βγ subunits (Gβγ) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the α-subunit of the Go protein. Go-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a Gβγ-sequestering peptide (βARK1ct). M1AChR and PAFR catalyzed Go α-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of Goα but not by similar mutants of Gi. Go-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to Go to activate a novel PKC-dependent mitogenic signaling pathway.

Authors
Biesen, TV; Hawes, BE; Raymond, JR; Luttrell, LM; Koch, WJ; Lefkowitz, RJ
MLA Citation
Biesen, TV, Hawes, BE, Raymond, JR, Luttrell, LM, Koch, WJ, and Lefkowitz, RJ. "Go-protein α-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism." Journal of Biological Chemistry 271.3 (1996): 1266-1269.
Source
scival
Published In
Journal of Biological Chemistry
Volume
271
Issue
3
Publish Date
1996
Start Page
1266
End Page
1269

Functional interaction between angiotensin ii and grk5 in cultured vascular smooth muscle cells (vsmc) and rat aorta

G protein receptor kinases (GRKs) are involved in receptor desensitization. In HEK293 cells overexpressing the angiotensin II (ang U) ATiA receptor (ATjAR) and GRK5, the ATjAR is a substrate for GRK5 and coupling to phospholipase C (PLC) is diminished. In the current study, we examined the interaction between ang II and GRKS in cultured VSMC from rat aorta, both in terms of functional consequences and long-term regulation. We stably transfected VSMC with a 1.8 kb fragment of bovine antisense GRK5, and measured GRK5 mRNA and protein expression, as well as ang n activation of PLC and phospholipase D (PLD). In antisense transfected cells, GRK5 mRNA was reduced by 64%. Both PLC and PLD activity, as measured by [3H]inositol and [3H]choline release, were increased (129±19% and 144±14% nontransfected levels, respectively), suggesting that GRK5 may be involved in the normal termination of VSMC ATi A& signaling. We next examined ang II regulation of GRKS expression. In cultured VSMC, GRK5 mRNA and protein is upregulated by exposure to 100 nM ang n for 16 h (453±27%). In aortas from rats receiving continuous infusion of ang II (0.7mg/ kg/day), GRK5 is also upregulated after 5 days (303±20%). This upregulation is completely blocked by the ATiAR antagonist losartan, and partially by the nonspecific vasodilator hydralazine, implying that GRKS may be regulated both by ang II and elevated blood pressure. In conclusion, GRKS appears to modulate AT| AR signaling in VSMC, and is itself regulated by ang II, suggesting a novel mechanism for regulating VSMC sensitivity to ang H.

Authors
Ishizaka, N; Griendling, K; Fukui, T; Oppermann, M; Lefkowitz, R; Alexander, R
MLA Citation
Ishizaka, N, Griendling, K, Fukui, T, Oppermann, M, Lefkowitz, R, and Alexander, R. "Functional interaction between angiotensin ii and grk5 in cultured vascular smooth muscle cells (vsmc) and rat aorta." FASEB Journal 10.6 (1996): A1134-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
6
Publish Date
1996
Start Page
A1134

Monoclonal antibodies reveal receptor specificity among G protein-coupled receptor kinases.

Authors
Oppermann, M; Kirchhoff, S; Lefkowitz, RJ
MLA Citation
Oppermann, M, Kirchhoff, S, and Lefkowitz, RJ. "Monoclonal antibodies reveal receptor specificity among G protein-coupled receptor kinases." JOURNAL OF LEUKOCYTE BIOLOGY (1996): 203-203.
Source
wos-lite
Published In
Journal of leukocyte biology
Publish Date
1996
Start Page
203
End Page
203

Monoclonal antibodies reveal receptor specificity among G protein-coupled receptor kinases

Authors
Oppermann, M; Kirchhoff, S; Lefkowitz, RJ
MLA Citation
Oppermann, M, Kirchhoff, S, and Lefkowitz, RJ. "Monoclonal antibodies reveal receptor specificity among G protein-coupled receptor kinases." JOURNAL OF LEUKOCYTE BIOLOGY (1996): 205-205.
Source
wos-lite
Published In
Journal of leukocyte biology
Publish Date
1996
Start Page
205
End Page
205

Recent insights into the mechanisms of agonist-specific G protein-coupled receptor desensitization and resensitization

Authors
Pitcher, JA; Krueger, KM; Touhara, K; Lefkowitz, RJ
MLA Citation
Pitcher, JA, Krueger, KM, Touhara, K, and Lefkowitz, RJ. "Recent insights into the mechanisms of agonist-specific G protein-coupled receptor desensitization and resensitization." 1996.
Source
wos-lite
Published In
Alfred Benzon Symposium
Volume
39
Publish Date
1996
Start Page
91
End Page
104

Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity.

To obtain large quantities of pure human beta 2-adrenergic receptor (beta 2-AR) needed for structural studies, an efficient method for beta 2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged beta 2-AR was expressed in Sf9 cells with a specific activity of 5-20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-beta-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60-70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of > 30%. The purified receptor was concentrated to > 1 mg/ml without loss of ligand binding activity.

Authors
Kwatra, MM; Schreurs, J; Schwinn, DA; Innis, MA; Caron, MG; Lefkowitz, RJ
MLA Citation
Kwatra, MM, Schreurs, J, Schwinn, DA, Innis, MA, Caron, MG, and Lefkowitz, RJ. "Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity." Protein Expr Purif 6.6 (December 1995): 717-721.
PMID