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Shenoy, Sudha Kaup

Positions:

Associate Professor in Medicine

Medicine, Cardiology
School of Medicine

Associate Professor in Cell Biology

Cell Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1998

Ph.D. — Oklahoma State University at Stillwater

Grants:

Multidisciplinary Heart and Vascular Diseases

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
March 31, 2021

Establishing the molecular and cellular mechanisms of Lgr5 signaling for controlling cancer stem cell behavior

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 01, 2017
End Date
August 31, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Paracrine Control of Blood Pressure by Renal Intercalated Cells

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
May 19, 2015
End Date
April 30, 2020

Regulation of B-arrestin2's pro-atherogenic activity by the deubiquitinase USP20

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 03, 2014
End Date
December 31, 2018

E3 Ligases and Deubiquitinases in GPCR Down Regulation

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2005
End Date
June 30, 2016

B-Arrestins and G Protein-Coupled Receptor Kinases in Cardiovascular Function

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Research Professional
Start Date
June 01, 2002
End Date
June 30, 2012
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Publications:

G Protein-Coupled Receptor Signaling Through β-Arrestin-Dependent Mechanisms.

β-arrestin1 (or arrestin2) and β-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that β-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by β-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted β-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that β-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by β-arrestins and highlight the physiologic effects of β-arrestin-dependent signaling.

Authors
Jean-Charles, P-Y; Kaur, S; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Kaur, S, and Shenoy, SK. "G Protein-Coupled Receptor Signaling Through β-Arrestin-Dependent Mechanisms." Journal of cardiovascular pharmacology 70.3 (September 2017): 142-158.
PMID
28328745
Source
epmc
Published In
Journal of Cardiovascular Pharmacology
Volume
70
Issue
3
Publish Date
2017
Start Page
142
End Page
158
DOI
10.1097/fjc.0000000000000482

Regulation of inflammation by β-arrestins: Not just receptor tales.

The ubiquitously expressed, multifunctional scaffolding proteins β-arrestin1 and β-arrestin2 each affect inflammatory signaling in a variety of cell lines. In addition to binding the carboxyl-terminal tails of innumerable 7-transmembrane receptors, β-arrestins scaffold untold numbers of other plasma membrane and cytoplasmic proteins. Consequently, the effects of β-arrestins on inflammatory signaling are diverse, and context-specific. This review highlights the roles of β-arrestins in regulating canonical activation of the pro-inflammatory transcription factor NFκB.

Authors
Freedman, NJ; Shenoy, SK
MLA Citation
Freedman, NJ, and Shenoy, SK. "Regulation of inflammation by β-arrestins: Not just receptor tales." Cellular signalling (February 9, 2017). (Review)
PMID
28189586
Source
epmc
Published In
Cellular Signalling
Publish Date
2017
DOI
10.1016/j.cellsig.2017.02.008

GPCR desensitization: Acute and prolonged phases.

G protein-coupled receptors (GPCRs) transduce a wide array of extracellular signals and regulate virtually every aspect of physiology. While GPCR signaling is essential, overstimulation can be deleterious, resulting in cellular toxicity or uncontrolled cellular growth. Accordingly, nature has developed a number of mechanisms for limiting GPCR signaling, which are broadly referred to as desensitization, and refer to a decrease in response to repeated or continuous stimulation. Short-term desensitization occurs over minutes, and is primarily associated with β-arrestins preventing G protein interaction with a GPCR. Longer-term desensitization, referred to as downregulation, occurs over hours to days, and involves receptor internalization into vesicles, degradation in lysosomes and decreased receptor mRNA levels through unclear mechanisms. Phosphorylation of the receptor by GPCR kinases (GRKs) and the recruitment of β-arrestins is critical to both these short- and long-term desensitization mechanisms. In addition to phosphorylation, both the GPCR and β-arrestins are modified post-translationally in several ways, including by ubiquitination. For many GPCRs, receptor ubiquitination promotes degradation of agonist-activated receptors in the lysosomes. Other proteins also play important roles in desensitization, including phosphodiesterases, RGS family proteins and A-kinase-anchoring proteins. Together, this intricate network of kinases, ubiquitin ligases, and adaptor proteins orchestrate the acute and prolonged desensitization of GPCRs.

Authors
Rajagopal, S; Shenoy, SK
MLA Citation
Rajagopal, S, and Shenoy, SK. "GPCR desensitization: Acute and prolonged phases." Cellular signalling (January 27, 2017). (Review)
PMID
28137506
Source
epmc
Published In
Cellular Signalling
Publish Date
2017
DOI
10.1016/j.cellsig.2017.01.024

Beta(1)-Adrenergic Receptor-induced Cardiac Contractility is dependent on MDM2-mediated GRK2 Turnover

Authors
Jean-Charles, P-Y; Yu, SM-W; Mao, L; Kommadi, RP; Zhang, Z; Stiber, J; Bowles, D; Jones, S; Abraham, D; Koch, WJ; Rockman, HA; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Yu, SM-W, Mao, L, Kommadi, RP, Zhang, Z, Stiber, J, Bowles, D, Jones, S, Abraham, D, Koch, WJ, Rockman, HA, and Shenoy, SK. "Beta(1)-Adrenergic Receptor-induced Cardiac Contractility is dependent on MDM2-mediated GRK2 Turnover." December 9, 2016.
Source
wos-lite
Published In
Circulation Research
Volume
119
Issue
12
Publish Date
2016
Start Page
E162
End Page
E163

Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.

Authors
Jean-Charles, P-Y; Rajiv, V; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Rajiv, V, and Shenoy, SK. "Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction." Journal of cellular physiology 231.10 (October 2016): 2071-2080. (Review)
PMID
26790995
Source
epmc
Published In
Journal of Cellular Physiology
Volume
231
Issue
10
Publish Date
2016
Start Page
2071
End Page
2080
DOI
10.1002/jcp.25317

Cover Image, Volume 231, Number 10, October 2016.

Cover: The cover image, by Sudha K. Shenoy et al., is based on the Mini-Review Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction, DOI: 10.1002/jcp.25317.

Authors
Jean-Charles, P-Y; Rajiv, V; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Rajiv, V, and Shenoy, SK. "Cover Image, Volume 231, Number 10, October 2016." Journal of cellular physiology 231.10 (October 2016): i-.
PMID
27315163
Source
epmc
Published In
Journal of Cellular Physiology
Volume
231
Issue
10
Publish Date
2016
Start Page
i
DOI
10.1002/jcp.25462

Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection.

On Earth, biological systems have evolved in response to environmental stressors, interactions dictated by physical forces that include gravity. The absence of gravity is an extreme stressor and the impact of its absence on biological systems is ill-defined. Astronauts who have spent extended time under conditions of minimal gravity (microgravity) experience an array of biological alterations, including perturbations in cardiovascular function. We hypothesized that physiological perturbations in cardiac function in microgravity may be a consequence of alterations in molecular and organellar dynamics within the cellular milieu of cardiomyocytes. We used a combination of mass spectrometry-based approaches to compare the relative abundance and turnover rates of 848 and 196 proteins, respectively, in rat neonatal cardiomyocytes exposed to simulated microgravity or normal gravity. Gene functional enrichment analysis of these data suggested that the protein content and function of the mitochondria, ribosomes, and endoplasmic reticulum were differentially modulated in microgravity. We confirmed experimentally that in microgravity protein synthesis was decreased while apoptosis, cell viability, and protein degradation were largely unaffected. These data support our conclusion that in microgravity cardiomyocytes attempt to maintain mitochondrial homeostasis at the expense of protein synthesis. The overall response to this stress may culminate in cardiac muscle atrophy.

Authors
Feger, BJ; Thompson, JW; Dubois, LG; Kommaddi, RP; Foster, MW; Mishra, R; Shenoy, SK; Shibata, Y; Kidane, YH; Moseley, MA; Carnell, LS; Bowles, DE
MLA Citation
Feger, BJ, Thompson, JW, Dubois, LG, Kommaddi, RP, Foster, MW, Mishra, R, Shenoy, SK, Shibata, Y, Kidane, YH, Moseley, MA, Carnell, LS, and Bowles, DE. "Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection." Scientific Reports 6 (September 27, 2016): 34091-.
PMID
27670941
Source
epmc
Published In
Scientific Reports
Volume
6
Publish Date
2016
Start Page
34091
DOI
10.1038/srep34091

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation.

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein β-arrestin2. Although β-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic β-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of β-arrestin2 activity could be dictated by β-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were β-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that β-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with β-arrestin2 was augmented when β-arrestin2 ubiquitination was prevented and reduced when β-arrestin2 ubiquitination was rendered constitutive. Constitutive β-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of β-arrestin2 are determined by β-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of β-arrestin2.

Authors
Jean-Charles, P-Y; Zhang, L; Wu, J-H; Han, S-O; Brian, L; Freedman, NJ; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Zhang, L, Wu, J-H, Han, S-O, Brian, L, Freedman, NJ, and Shenoy, SK. "Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation." The Journal of biological chemistry 291.14 (April 2016): 7450-7464.
PMID
26839314
Source
epmc
Published In
The Journal of biological chemistry
Volume
291
Issue
14
Publish Date
2016
Start Page
7450
End Page
7464
DOI
10.1074/jbc.m115.687129

Chapter Nine - Cellular Roles of Beta-Arrestins as Substrates and Adaptors of Ubiquitination and Deubiquitination.

β-Arrestin1 and β-arrestin2 are homologous adaptor proteins that are ubiquitously expressed in mammalian cells. They belong to a four-member family of arrestins that regulate the vast family of seven-transmembrane receptors that couple to heterotrimeric G proteins (7TMRs or GPCRs), and that modulate 7TMR signal transduction. β-Arrestins were originally identified in the context of signal inhibition via the 7TMRs because they competed with and thereby blocked G protein coupling to 7TMRs. Currently, in addition to their role as desensitizers of signaling, β-arrestins are appreciated as multifunctional adaptors that mediate trafficking and signal transduction of not only 7TMRs, but a growing list of additional receptors, ion channels, and nonreceptor proteins. β-Arrestins' interactions with their multifarious partners are based on their dynamic conformational states rather than particular domain-domain interactions. β-Arrestins adopt activated conformations upon 7TMR association. In addition, β-arrestins undergo various posttranslational modifications that are choreographed by activated 7TMRs, including phosphorylation, ubiquitination, acetylation, nitrosylation, and SUMOylation. Ubiquitination of β-arrestins is critical for their high-affinity interaction with 7TMRs as well as with endocytic adaptor proteins and signaling kinases. β-Arrestins also function as critical adaptors for ubiquitination and deubiquitination of various cellular proteins, and thereby affect the longevity of signal transducers and the intensity of signal transmission.

Authors
Jean-Charles, P-Y; Freedman, NJ; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Freedman, NJ, and Shenoy, SK. "Chapter Nine - Cellular Roles of Beta-Arrestins as Substrates and Adaptors of Ubiquitination and Deubiquitination." Progress in molecular biology and translational science 141 (January 2016): 339-369. (Review)
PMID
27378762
Source
epmc
Published In
Progress in Molecular Biology and Translational Science
Volume
141
Publish Date
2016
Start Page
339
End Page
369
DOI
10.1016/bs.pmbts.2016.04.003

Preface.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "Preface." Progress in molecular biology and translational science 141 (January 2016): xiii-xiv.
PMID
27378763
Source
epmc
Published In
Progress in Molecular Biology and Translational Science
Volume
141
Publish Date
2016
Start Page
xiii
End Page
xiv
DOI
10.1016/s1877-1173(16)30035-7

Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors.

The seven-transmembrane containing G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. Transmembrane signaling by GPCRs is fundamental to many aspects of physiology including vision, olfaction, cardiovascular, and reproductive functions as well as pain, behavior and psychomotor responses. The duration and magnitude of signal transduction is tightly controlled by a series of coordinated trafficking events that regulate the cell-surface expression of GPCRs at the plasma membrane. Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs. Of the various molecular mechanisms that impart selectivity, sensitivity and strength of transmembrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR. Ubiquitination of proteins was originally discovered in the context of lysosome-independent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes. In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling. To date about 40 GPCRs are known to be ubiquitinated. For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes. This chapter will focus on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

Authors
Jean-Charles, P-Y; Snyder, JC; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Snyder, JC, and Shenoy, SK. "Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors." Progress in molecular biology and translational science 141 (January 2016): 1-55. (Review)
PMID
27378754
Source
epmc
Published In
Progress in Molecular Biology and Translational Science
Volume
141
Publish Date
2016
Start Page
1
End Page
55
DOI
10.1016/bs.pmbts.2016.05.001

PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE Ubiquitination and Transmembrane Signaling

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE Ubiquitination and Transmembrane Signaling." UBIQUITINATION AND TRANSMEMBRANE SIGNALING 141 (2016): XIII-XIV.
Source
wos-lite
Published In
Progress in Molecular Biology and Translational Science
Volume
141
Publish Date
2016
Start Page
XIII
End Page
XIV

Pacemaker Induced Transient Asynchrony (PITA) Restores Contractile Reserve in Synchronous Heart Failure

Authors
Kirk, JA; Chakir, K; Lee, K; Pironti, G; Ranek, MJ; Tunin, RS; de Tombe, P; Shenoy, SK; Rockman, HA; Craig, R; Kass, DA
MLA Citation
Kirk, JA, Chakir, K, Lee, K, Pironti, G, Ranek, MJ, Tunin, RS, de Tombe, P, Shenoy, SK, Rockman, HA, Craig, R, and Kass, DA. "Pacemaker Induced Transient Asynchrony (PITA) Restores Contractile Reserve in Synchronous Heart Failure." July 17, 2015.
Source
wos-lite
Published In
Circulation Research
Volume
117
Publish Date
2015

Phosphorylation of the deubiquitinase USP20 by protein kinase A regulates post-endocytic trafficking of β2 adrenergic receptors to autophagosomes during physiological stress.

Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.

Authors
Kommaddi, RP; Jean-Charles, P-Y; Shenoy, SK
MLA Citation
Kommaddi, RP, Jean-Charles, P-Y, and Shenoy, SK. "Phosphorylation of the deubiquitinase USP20 by protein kinase A regulates post-endocytic trafficking of β2 adrenergic receptors to autophagosomes during physiological stress." The Journal of biological chemistry 290.14 (April 2015): 8888-8903.
PMID
25666616
Source
epmc
Published In
The Journal of biological chemistry
Volume
290
Issue
14
Publish Date
2015
Start Page
8888
End Page
8903
DOI
10.1074/jbc.m114.630541

Arrestin interaction with E3 ubiquitin ligases and deubiquitinases: functional and therapeutic implications.

Arrestins constitute a small family of four homologous adaptor proteins (arrestins 1-4), which were originally identified as inhibitors of signal transduction elicited by the seven-transmembrane G protein-coupled receptors. Currently arrestins (especially arrestin2 and arrestin3; also called β-arrestin1 and β-arrestin2) are known to be activators of cell signaling and modulators of endocytic trafficking. Arrestins mediate these effects by binding to not only diverse cell-surface receptors but also by associating with a variety of critical signaling molecules in different intracellular compartments. Thus, the functions of arrestins are multifaceted and demand interactions with a host of proteins and require an array of selective conformations. Furthermore, receptor ligands that specifically induce signaling via arrestins are being discovered and their physiological roles are emerging. Recent evidence suggests that the activity of arrestin is regulated in space and time by virtue of its dynamic association with specific enzymes of the ubiquitination pathway. Ubiquitin-dependent, arrestin-mediated signaling could serve as a potential platform for developing novel therapeutic strategies to target transmembrane signaling and physiological responses.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "Arrestin interaction with E3 ubiquitin ligases and deubiquitinases: functional and therapeutic implications." Handb Exp Pharmacol 219 (2014): 187-203.
PMID
24292831
Source
pubmed
Published In
Handbook of experimental pharmacology
Volume
219
Publish Date
2014
Start Page
187
End Page
203
DOI
10.1007/978-3-642-41199-1_10

Deubiquitinases and their emerging roles in β-arrestin-mediated signaling.

The two homologous mammalian proteins called β-arrestin1 (also known as arrestin2) and β-arrestin2 (also called arrestin3) are now widely accepted as endocytic and signaling adaptors for G protein-coupled receptors (GPCRs), growth factor receptors, and ion channels. The sustained interactions of β-arrestins with activated GPCRs have been shown to correlate with the agonist-induced ubiquitination on distinct domains in the β-arrestin molecule. Additionally, ubiquitination of β-arrestin promotes its interaction with proteins that mediate endocytosis (e.g., clathrin) and signaling (e.g., c-RAF). Recent studies have demonstrated that deubiquitination of β-arrestin by specific deubiquitinating enzymes (DUBs) acts as an important regulatory mechanism, which determines the stability of β-arrestin-GPCR binding and fine-tunes β-arrestin-dependent signaling to downstream kinases. Accordingly, ubiquitination/deubiquitination of β-arrestin can serve as an on/off switch for its signaling and endocytic functions. Moreover, by regulating the stability and localization of signalosomes, deubiquitination of β-arrestins by DUBs imparts spatial and temporal resolution in GPCR signaling.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "Deubiquitinases and their emerging roles in β-arrestin-mediated signaling." Methods Enzymol 535 (2014): 351-370.
PMID
24377933
Source
pubmed
Published In
Methods in Enzymology
Volume
535
Publish Date
2014
Start Page
351
End Page
370
DOI
10.1016/B978-0-12-397925-4.00020-1

Regulation of β2Adrenergic Receptor Trafficking and Signaling by Ubiquitination and Deubiquitination

Authors
Shenoy, S
MLA Citation
Shenoy, S. "Regulation of β2Adrenergic Receptor Trafficking and Signaling by Ubiquitination and Deubiquitination." (December 1, 2013): 95-96. (Chapter)
Source
scopus
Publish Date
2013
Start Page
95
End Page
96
DOI
10.1016/B978-0-12-800044-1.00082-9

Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking.

β-arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven-transmembrane receptor trafficking and signalling. Other proteins with predicted 'arrestin-like' structural domains but lacking sequence homology have been indicated to function like β-arrestin in receptor regulation. We demonstrate that β-arrestin2 is the primary adaptor that rapidly binds agonist-activated β(2) adrenergic receptors (β(2)ARs) and promotes clathrin-dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin-dependent lysosomal degradation of the receptor. The arrestin-domain-containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β(2)AR-Nedd4 complexes on endosomes and do not affect the adaptor roles of β-arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4-β(2)AR complexes to a subpopulation of early endosomes.

Authors
Han, S-O; Kommaddi, RP; Shenoy, SK
MLA Citation
Han, S-O, Kommaddi, RP, and Shenoy, SK. "Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking." EMBO Rep 14.2 (February 2013): 164-171.
PMID
23208550
Source
pubmed
Published In
EMBO Reports
Volume
14
Issue
2
Publish Date
2013
Start Page
164
End Page
171
DOI
10.1038/embor.2012.187

Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking

β-arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven-transmembrane receptor trafficking and signalling. Other proteins with predicted 'arrestin-like' structural domains but lacking sequence homology have been indicated to function like β-arrestin in receptor regulation. We demonstrate that β-arrestin2 is the primary adaptor that rapidly binds agonist-activated β2 adrenergic receptors (β2 ARs) and promotes clathrin-dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin-dependent lysosomal degradation of the receptor. The arrestin-domain-containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β2 AR-Nedd4 complexes on endosomes and do not affect the adaptor roles of β-arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4-β2 AR complexes to a subpopulation of early endosomes. © 2013 European Molecular Biology Organization.

Authors
Han, S-O; Kommaddi, RP; Shenoy, SK
MLA Citation
Han, S-O, Kommaddi, RP, and Shenoy, SK. "Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking." EMBO Reports 14.2 (2013): 164-171.
Source
scival
Published In
EMBO Reports
Volume
14
Issue
2
Publish Date
2013
Start Page
164
End Page
171
DOI
10.1038/embor.2012.187

Arrestins and protein ubiquitination

The adaptor proteins, β-arrestins 1 and 2, were originally identified as inhibitors of G protein signaling at the seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors or GPCRs). Subsequent studies have established β-arrestins as critical multifunctional 7TMR adaptors that mediate receptor trafficking and activate G protein-independent signaling pathways. 7TMR activation leads not only to the recruitment of arrestin proteins upon phosphorylation by GPCR kinases but also to β-arrestin ubiquitination. This posttranslational modification of β-arrestin is appended by specific E3 ubiquitin ligases and reversed by deubiquitinases, which are also recruited in a receptor- and agonist-specific manner. β-Arrestin ubiquitination allows it to form protein complexes with activated 7TMRs, endocytic proteins such as clathrin, and phosphorylated ERK1/2. β-Arrestin ubiquitination is dependent on its activated conformation and likely regulates timing and subcellular localization of various protein interactions during receptor trafficking and signaling. β-Arrestins also serve as adaptors that escort E3 ubiquitin ligases to mediate ubiquitination of a wide list of substrate proteins including 7TMRs and provide an added layer of regulation for defining substrate specificity in the cellular ubiquitination pathway. © 2013 Elsevier Inc.

Authors
Kommaddi, RP; Shenoy, SK
MLA Citation
Kommaddi, RP, and Shenoy, SK. "Arrestins and protein ubiquitination." Progress in Molecular Biology and Translational Science 118 (2013): 175-204.
PMID
23764054
Source
scival
Published In
Progress in Molecular Biology and Translational Science
Volume
118
Publish Date
2013
Start Page
175
End Page
204
DOI
10.1016/B978-0-12-394440-5.00007-3

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β(2)-adrenergic receptors.

Lysosomal degradation of ubiquitinated β(2)-adrenergic receptors (β(2)ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the βAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed β(2)ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-β(2)ARs in HEK-293 cells. Carvedilol prevented β(2)AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography-tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound β(2)AR. The association of MARCH2 with internalized β(2)ARs was stabilized by carvedilol and did not involve β-arrestin. Small interfering RNA-mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous β(2)ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β(2)AR expression and, consequently, its signaling.

Authors
Han, S-O; Xiao, K; Kim, J; Wu, J-H; Wisler, JW; Nakamura, N; Freedman, NJ; Shenoy, SK
MLA Citation
Han, S-O, Xiao, K, Kim, J, Wu, J-H, Wisler, JW, Nakamura, N, Freedman, NJ, and Shenoy, SK. "MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β(2)-adrenergic receptors." J Cell Biol 199.5 (November 26, 2012): 817-830.
PMID
23166351
Source
pubmed
Published In
The Journal of Cell Biology
Volume
199
Issue
5
Publish Date
2012
Start Page
817
End Page
830
DOI
10.1083/jcb.201208192

beta-arrestin2 and ARRDC proteins have distinct roles in beta(2)AR trafficking and signaling

Authors
Han, S-O; Kommaddi, RP; Venkataramanan, V; Shenoy, SK
MLA Citation
Han, S-O, Kommaddi, RP, Venkataramanan, V, and Shenoy, SK. "beta-arrestin2 and ARRDC proteins have distinct roles in beta(2)AR trafficking and signaling." April 2012.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
26
Publish Date
2012

G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors.

OBJECTIVE: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. METHODS AND RESULTS: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe(-/-) mice, carotid interposition grafts from Grk5(-/-) mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5(-/-) with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-β in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-κB-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-κB activity in macrophages. CONCLUSIONS: GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-κB activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-β.

Authors
Wu, J-H; Zhang, L; Fanaroff, AC; Cai, X; Sharma, KC; Brian, L; Exum, ST; Shenoy, SK; Peppel, K; Freedman, NJ
MLA Citation
Wu, J-H, Zhang, L, Fanaroff, AC, Cai, X, Sharma, KC, Brian, L, Exum, ST, Shenoy, SK, Peppel, K, and Freedman, NJ. "G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors." Arterioscler Thromb Vasc Biol 32.2 (February 2012): 308-316.
PMID
22095977
Source
pubmed
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
32
Issue
2
Publish Date
2012
Start Page
308
End Page
316
DOI
10.1161/ATVBAHA.111.239608

β-arrestin1 mediates metastatic growth of breast cancer cells by facilitating HIF-1-dependent VEGF expression.

β-Arrestins 1 and 2 are multifunctional adaptor proteins originally discovered for their role in desensitizing seven-transmembrane receptor signaling via the heterotrimeric guanine nucleotide-binding proteins. Recently identified roles of β-arrestins include regulation of cancer cell chemotaxis and proliferation. Herein, we report that β-arrestin1 expression regulates breast tumor colonization in nude mice and cancer cell viability during hypoxia. β-Arrestin1 robustly interacts with nuclear hypoxia-induced factor-1α (HIF-1α) that is stabilized during hypoxia and potentiates HIF-1-dependent transcription of the angiogenic factor vascular endothelial growth factor-A (VEGF-A). Increased expression of β-arrestin1 in human breast cancer (infiltrating ductal carcinoma or IDC and metastatic IDC) correlates with increased levels of VEGF-A. While the anti-angiogenic drug thalidomide inhibits HIF-1-dependent VEGF transcription in breast carcinoma cells, it does not prevent HIF-1α stabilization, but leads to aberrant localization of HIF-1α to the perinuclear compartments and surprisingly stimulates nuclear export of β-arrestin1. Additionally, imatinib mesylate that inhibits release of VEGF induces nuclear export of β-arrestin1-HIF-1α complexes. Our findings suggest that β-arrestin1 regulates nuclear signaling during hypoxia to promote survival of breast cancer cells via VEGF signaling and that drugs that induce its translocation from the nucleus to the cytoplasm could be useful in anti-angiogenic and breast cancer therapies.

Authors
Shenoy, SK; Han, S; Zhao, YL; Hara, MR; Oliver, T; Cao, Y; Dewhirst, MW
MLA Citation
Shenoy, SK, Han, S, Zhao, YL, Hara, MR, Oliver, T, Cao, Y, and Dewhirst, MW. "β-arrestin1 mediates metastatic growth of breast cancer cells by facilitating HIF-1-dependent VEGF expression." Oncogene 31.3 (January 19, 2012): 282-292.
PMID
21685944
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
31
Issue
3
Publish Date
2012
Start Page
282
End Page
292
DOI
10.1038/onc.2011.238

β-Arrestin and dishevelled coordinate biased signaling.

Authors
Schulte, G; Shenoy, SK
MLA Citation
Schulte, G, and Shenoy, SK. "β-Arrestin and dishevelled coordinate biased signaling." Proc Natl Acad Sci U S A 108.50 (December 13, 2011): 19839-19840.
PMID
22123954
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
50
Publish Date
2011
Start Page
19839
End Page
19840
DOI
10.1073/pnas.1117444108

Cardiovascular biology: heart fails without pump partner.

Authors
Shenoy, SK; Rockman, HA
MLA Citation
Shenoy, SK, and Rockman, HA. "Cardiovascular biology: heart fails without pump partner. (Published online)" Nature 477.7366 (September 28, 2011): 546-547.
PMID
21956327
Source
pubmed
Published In
Nature
Volume
477
Issue
7366
Publish Date
2011
Start Page
546
End Page
547
DOI
10.1038/477546a

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

A tale of two sites: How ubiquitination of a G protein-coupled receptor is coupled to its lysosomal trafficking from distinct receptor domains.

The β(2)-adrenergic receptor (β(2)AR) is a prototypical G(s)-coupled receptor belonging to the superfamily of seven transmembrane spanning heptahelical receptors (7TMRs or G protein-coupled receptors [GPCRs])-therapeutically the most diverse and accessible class of cell surface receptors. The classic pathway of β(2)AR signaling (Fig. 1) is triggered by activation of the heterotrimeric G protein G(s) by agonists (catecholamines-noradrenaline and adrenaline). This in turn activates adenylyl cyclase leading to the generation of second messenger signaling molecules (cyclic adenosine monophosphates, cAMP) which subsequently activate protein kinase A (PKA) as well as some ion channels, such as the class C type of L-type calcium channels, Ca(v)1.2.31 Here in we review how trafficking and signaling of the β(2)AR is regulated by the post-translational modification, ubiquitination.1.

Authors
Sarker, S; Xiao, K; Shenoy, SK
MLA Citation
Sarker, S, Xiao, K, and Shenoy, SK. "A tale of two sites: How ubiquitination of a G protein-coupled receptor is coupled to its lysosomal trafficking from distinct receptor domains." Commun Integr Biol 4.5 (September 2011): 528-531.
PMID
22046454
Source
pubmed
Published In
Communicative & integrative biology
Volume
4
Issue
5
Publish Date
2011
Start Page
528
End Page
531
DOI
10.4161/cib.4.5.16458

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

Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains.

Agonist stimulation of the β2-adrenergic receptors (β2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β2ARs in nonlysosomal compartments. Additionally, a lysine-less β2AR (0K-β2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β2AR. However, only loop 3 and carboxyl tail β2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β2AR. We overexpressed and purified the β2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved β2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β2AR. These findings introduce a new concept that two distinct domains in the β2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.

Authors
Xiao, K; Shenoy, SK
MLA Citation
Xiao, K, and Shenoy, SK. "Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains." J Biol Chem 286.14 (April 8, 2011): 12785-12795.
PMID
21330366
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
14
Publish Date
2011
Start Page
12785
End Page
12795
DOI
10.1074/jbc.M110.203091

β2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains

Agonist stimulation of the β 2 -adrenergic receptors (β 2 ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β 2 ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β 2 ARs in nonlysosomal compartments. Additionally, a lysineless β 2 AR(0K-β 2 AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β 2 AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β 2 ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β 2 AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β 2 AR. However, only loop 3 and carboxyl tail β 2 ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β 2 AR. We overexpressed and purified the β 2 AR from HEK-293 cells with or without prior agonist exposure and subjected trypsincleaved β 2 AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β 2 AR. These findings introduce a new concept that two distinct domains in the β 2 AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Xiao, K; Shenoy, SK
MLA Citation
Xiao, K, and Shenoy, SK. "β2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains." Journal of Biological Chemistry 286.14 (April 8, 2011): 12785-12795.
Source
scopus
Published In
The Journal of biological chemistry
Volume
286
Issue
14
Publish Date
2011
Start Page
12785
End Page
12795
DOI
10.1074/jbc.M110.203091

β-arrestin-biased signaling by the β-adrenergic receptors.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "β-arrestin-biased signaling by the β-adrenergic receptors." Curr Top Membr 67 (2011): 51-78. (Review)
PMID
21771485
Source
pubmed
Published In
Current Topis in Membranes
Volume
67
Publish Date
2011
Start Page
51
End Page
78
DOI
10.1016/B978-0-12-384921-2.00003-3

Visualizing G protein-coupled receptor signalsomes using confocal immunofluorescence microscopy.

The heptahelical G protein-coupled receptors (GPCRs) receive and transmit a wide range of extracellular stimuli and induce a wide array of cellular responses by activating signaling kinases. It has become increasingly evident that the agonist-stimulated GPCR complexed with the adaptor protein, β-arrestin, serves as a focal point to recruit, activate, and target kinases to discrete subcellular compartments. This chapter describes a protocol to visualize the changes in the subcellular distribution of activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) when induced by the angiotensin II type 1a receptor.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "Visualizing G protein-coupled receptor signalsomes using confocal immunofluorescence microscopy." Methods Mol Biol 756 (2011): 333-342.
PMID
21870237
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
756
Publish Date
2011
Start Page
333
End Page
342
DOI
10.1007/978-1-61779-160-4_20

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

beta-arrestin2 and ARRDC proteins have distinct roles in beta(2)AR trafficking and signaling

Authors
Han, S-O; Kommaddi, RP; Venkataramanan, V; Shenoy, SK
MLA Citation
Han, S-O, Kommaddi, RP, Venkataramanan, V, and Shenoy, SK. "beta-arrestin2 and ARRDC proteins have distinct roles in beta(2)AR trafficking and signaling." FASEB JOURNAL 24 (April 2010).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
24
Publish Date
2010

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

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Authors
Berthouze, M; Venkataramanan, V; Li, Y; Shenoy, SK
MLA Citation
Berthouze, M, Venkataramanan, V, Li, Y, and Shenoy, SK. "The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization." EMBO J 28.12 (June 17, 2009): 1684-1696.
PMID
19424180
Source
pubmed
Published In
EMBO Journal
Volume
28
Issue
12
Publish Date
2009
Start Page
1684
End Page
1696
DOI
10.1038/emboj.2009.128

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

Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis.

Signaling by the platelet-derived growth factor receptor-beta (PDGFRbeta) is diminished when the PDGFRbeta is phosphorylated on seryl residues by G protein-coupled receptor kinase-5 (GRK5), but mechanisms for GRK5 activation by the PDGFRbeta remain obscure. We therefore tested whether the PDGFRbeta is able to tyrosine-phosphorylate and thereby activate GRK5. Purified GRK5 was tyrosine-phosphorylated by the wild-type PDGFRbeta to a stoichiometry of 0.8 mol phosphate/mol GRK5, an extent approximately 5 times greater than observed with a Y857F PDGFRbeta mutant that fails to phosphorylate exogenous substrates but autophosphorylates and activates Src normally. The degree of PDGFRbeta-mediated phosphorylation of GRK5 correlated with GRK5 activity, as assessed by seryl phosphorylation of the PDGFRbeta in purified protein preparations, in intact cells expressing a tyrosine-to-phenylalanine GRK5 mutant, and in GRK5 peptide phosphorylation assays. However, tyrosyl phosphorylation of GRK5 was not necessary for GRK5-mediated phosphorylation of the beta(2)-adrenergic receptor, even though beta(2)-adrenergic receptor activation promoted tyrosyl phosphorylation of GRK5 in smooth muscle cells. Phosphorylation of the PDGFRbeta by GRK5 in smooth muscle cells or in purified protein preparations reduced PDGFRbeta-mediated peptide phosphorylation. In contrast, phosphorylation of GRK5 by the PDGFRbeta enhanced the V(max) of GRK5-mediated peptide phosphorylation, by 3.4-fold, without altering the GRK5 K(M) for peptide. We conclude that GRK5 tyrosyl phosphorylation is required for the activation of GRK5 by the PDGFRbeta, but not by the beta(2)-adrenergic receptor, and that by activating GRK5, the PDGFRbeta triggers its own desensitization.

Authors
Cai, X; Wu, J-H; Exum, ST; Oppermann, M; Premont, RT; Shenoy, SK; Freedman, NJ
MLA Citation
Cai, X, Wu, J-H, Exum, ST, Oppermann, M, Premont, RT, Shenoy, SK, and Freedman, NJ. "Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis." Mol Pharmacol 75.3 (March 2009): 626-636.
PMID
19092051
Source
pubmed
Published In
Molecular pharmacology
Volume
75
Issue
3
Publish Date
2009
Start Page
626
End Page
636
DOI
10.1124/mol.108.050278

Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor.

Agonist-stimulated beta(2)-adrenergic receptor (beta(2)AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the beta(2)AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited beta(2)AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, beta(2)AR as well as beta-arrestin2, the endocytic and signaling adaptor for the beta(2)AR, interact robustly with Nedd4 upon agonist stimulation. However, beta(2)AR-Nedd4 interaction is ablated when beta-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for beta-arrestin2 in mediating beta(2)AR ubiquitination. Notably, beta-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in beta(2)AR trafficking. Collectively, our findings indicate that the degradative fate of the beta(2)AR in the lysosomal compartments is dependent upon beta-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.

Authors
Shenoy, SK; Xiao, K; Venkataramanan, V; Snyder, PM; Freedman, NJ; Weissman, AM
MLA Citation
Shenoy, SK, Xiao, K, Venkataramanan, V, Snyder, PM, Freedman, NJ, and Weissman, AM. "Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor." J Biol Chem 283.32 (August 8, 2008): 22166-22176.
PMID
18544533
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
32
Publish Date
2008
Start Page
22166
End Page
22176
DOI
10.1074/jbc.M709668200

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-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

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

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

Seven-transmembrane receptors and ubiquitination.

Regulation of protein function by posttranslational modification plays an important role in many biological pathways. The most well known among such modifications is protein phosphorylation performed by highly specific protein kinases. In the past decade, however, covalent linkage of the low-molecular-weight protein ubiquitin to substrate proteins (protein ubiquitination) has proven to be yet another widely used mechanism of protein regulation playing a crucial role in virtually all aspects of cellular functions. This review highlights some of the recently discovered and provocative roles for ubiquitination in the regulation of the life cycle and signal transduction properties of 7-transmembrane receptors that serve to integrate many biological functions and play fundamental roles in cardiovascular homeostasis.

Authors
Shenoy, SK
MLA Citation
Shenoy, SK. "Seven-transmembrane receptors and ubiquitination." Circ Res 100.8 (April 27, 2007): 1142-1154. (Review)
PMID
17463329
Source
pubmed
Published In
Circulation Research
Volume
100
Issue
8
Publish Date
2007
Start Page
1142
End Page
1154
DOI
10.1161/01.RES.0000261939.88744.5a

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

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

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

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

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 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

{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

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

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

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

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

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

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

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

Multifaceted roles of β-arrestins in the regulation of seven-transmembrane-spanning receptor trafficking and signaling

Authors
SHENOY, S
MLA Citation
SHENOY, S. "Multifaceted roles of β-arrestins in the regulation of seven-transmembrane-spanning receptor trafficking and signaling." Biochem J. 375 (2003): 503-515.
Source
cinii-english
Published In
Biochem J.
Volume
375
Publish Date
2003
Start Page
503
End Page
515

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

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

Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex.

Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.

Authors
Deng, K; Shenoy, SK; Tso, SC; Yu, L; Yu, CA
MLA Citation
Deng, K, Shenoy, SK, Tso, SC, Yu, L, and Yu, CA. "Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex." J Biol Chem 276.9 (March 2, 2001): 6499-6505.
PMID
11073949
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
9
Publish Date
2001
Start Page
6499
End Page
6505
DOI
10.1074/jbc.M007128200

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

Authors
SHENOY, S
MLA Citation
SHENOY, S. "Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin." Science 294 (2001): 1307-1313.
Source
cinii-english
Published In
Science
Volume
294
Publish Date
2001
Start Page
1307
End Page
1313

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex.

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

Authors
Tso, SC; Shenoy, SK; Quinn, BN; Yu, L
MLA Citation
Tso, SC, Shenoy, SK, Quinn, BN, and Yu, L. "Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex." J Biol Chem 275.20 (May 19, 2000): 15287-15294.
PMID
10748084
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
20
Publish Date
2000
Start Page
15287
End Page
15294
DOI
10.1074/jbc.M907367199

Structural basis of multifunctional bovine mitochondrial cytochrome bc1 complex.

The mitochondrial cytochrome bc1 complex is a multifunctional membrane protein complex. It catalyzes electron transfer, proton translocation, peptide processing, and superoxide generation. Crystal structure data at 2.9 A resolution not only establishes the location of the redox centers and inhibitor binding sites, but also suggests a movement of the head domain of the iron-sulfur protein (ISP) during bc1 catalysis and inhibition of peptide-processing activity during complex maturation. The functional importance of the movement of extramembrane (head) domain of ISP in the bc1 complex is confirmed by analysis of the Rhodobacter sphaeroides bc1 complex mutants with increased rigidity in the ISP neck and by the determination of rate constants for acid/base-induced intramolecular electron transfer between [2Fe-2S] and heme c1 in native and inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovine heart mitochondrial bc1 complex by nonionic detergent at concentrations that inactivate electron transfer activity. This peptide-processing activity is shown to be associated with subunits I and II by cloning, overexpression and in vitro reconstitution. The superoxide-generation site of the cytochrome bc1 complex is located at reduced bL and Q*-. The reaction is membrane potential-, and cytochrome c-dependent.

Authors
Yu, CA; Tian, H; Zhang, L; Deng, KP; Shenoy, SK; Yu, L; Xia, D; Kim, H; Deisenhofer, J
MLA Citation
Yu, CA, Tian, H, Zhang, L, Deng, KP, Shenoy, SK, Yu, L, Xia, D, Kim, H, and Deisenhofer, J. "Structural basis of multifunctional bovine mitochondrial cytochrome bc1 complex." J Bioenerg Biomembr 31.3 (June 1999): 191-199. (Review)
PMID
10591525
Source
pubmed
Published In
Journal of Bioenergetics and Biomembranes
Volume
31
Issue
3
Publish Date
1999
Start Page
191
End Page
199

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex.

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.

Authors
Yu, L; Tso, SC; Shenoy, SK; Quinn, BN; Xia, D
MLA Citation
Yu, L, Tso, SC, Shenoy, SK, Quinn, BN, and Xia, D. "The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex." J Bioenerg Biomembr 31.3 (June 1999): 251-257. (Review)
PMID
10591531
Source
pubmed
Published In
Journal of Bioenergetics and Biomembranes
Volume
31
Issue
3
Publish Date
1999
Start Page
251
End Page
257

Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase.

The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3. The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent. Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine. QPs3 was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase. Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million. The recombinant QPs3 binds ubiquinone, causing a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state. Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone. Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride. Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92. Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride.

Authors
Shenoy, SK; Yu, L; Yu, CA
MLA Citation
Shenoy, SK, Yu, L, and Yu, CA. "Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase." J Biol Chem 274.13 (March 26, 1999): 8717-8722.
PMID
10085111
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
13
Publish Date
1999
Start Page
8717
End Page
8722

Cloning and functional expression of core proteins of bovine heart mitochondrial bc(1).

Authors
Shenoy, SK; Deng, KP; Yu, L; Yu, CA
MLA Citation
Shenoy, SK, Deng, KP, Yu, L, and Yu, CA. "Cloning and functional expression of core proteins of bovine heart mitochondrial bc(1)." BIOPHYSICAL JOURNAL 74.2 (February 1998): A197-A197.
Source
wos-lite
Published In
Biophysical Journal
Volume
74
Issue
2
Publish Date
1998
Start Page
A197
End Page
A197

Reactivation, cloning and overexpression of matrix processing peptidase of the cytochrome bc(1) complex of bovine heart mitochondria

Authors
Yu, CA; Shenoy, SK; Deng, KP; Zhang, L; Yu, L
MLA Citation
Yu, CA, Shenoy, SK, Deng, KP, Zhang, L, and Yu, L. "Reactivation, cloning and overexpression of matrix processing peptidase of the cytochrome bc(1) complex of bovine heart mitochondria." FASEB JOURNAL 11.9 (July 31, 1997): A1280-A1280.
Source
wos-lite
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
A1280
End Page
A1280

The smallest membrane anchoring subunit (QPs3) of bovine heart mitochondrial succinate-ubiquinone reductase. Cloning, sequencing, topology, and Q-binding domain.

The cDNA encoding the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate-ubiquinone reductase was cloned and sequenced. This cDNA is 1330 base pairs long with an open reading frame of 474 base pairs that encodes the 103 amino acid residues of mature QPs3 and a 55-amino acid residue presequence. The cDNA insert has an 820-base pair long 3'-untranslated region, including a poly(A) tail. The molecular mass of QPs3, deduced from the nucleotide sequence, is 10,989 Da. QPs3 is a very hydrophobic protein; the hydropathy plot of the amino acid sequence reveals three transmembrane helices. Previous photoaffinity labeling studies of succinate-ubiquinone reductase, using 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), identified QPs3 as one of the putative Q-binding proteins in this reductase. An azido-Q-linked peptide with a retention time of 66 min is obtained by high performance liquid chromatography of the chymotrypsin digest of carboxymethylated and succinylated [3H]azido-Q-labeled QPs3 purified from labeled succinate-ubiquinone reductase by a procedure involving phenyl-Sepharose 4B column chromatography, preparative SDS-polyacrylamide gel electrophoresis, and acetone precipitation. The amino acid sequence of this peptide is NH2-L-N-P-C-S-A-M-D-Y-COOH, corresponding to residues 29-37. The structure of QPs3 in the inner mitochondrial membrane is proposed based on the hydropathy profile of the amino acid sequence, on the predicted tendencies to form alpha-helices and beta-sheets, and on immunobinding of Fab' fragmenthorseradish peroxidase conjugates prepared from antibodies against two synthetic peptides, corresponding to the NH2 terminus region and the loop connecting helices 2 and 3 of QPs3, in mitoplasts and submitochondrial particles. The ubiquinone-binding domain in the proposed model of QPs3 is probably located at the end of transmembrane helix 1 toward the C-side of the mitochondrial inner membrane.

Authors
Shenoy, SK; Yu, L; Yu, CA
MLA Citation
Shenoy, SK, Yu, L, and Yu, CA. "The smallest membrane anchoring subunit (QPs3) of bovine heart mitochondrial succinate-ubiquinone reductase. Cloning, sequencing, topology, and Q-binding domain." J Biol Chem 272.28 (July 11, 1997): 17867-17872.
PMID
9211943
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
28
Publish Date
1997
Start Page
17867
End Page
17872

Expression and properties of QPs3 of mitochondrial succinate-Q reductase.

Authors
Shenoy, SK; Yu, L; Yu, CA
MLA Citation
Shenoy, SK, Yu, L, and Yu, CA. "Expression and properties of QPs3 of mitochondrial succinate-Q reductase." BIOPHYSICAL JOURNAL 72.2 (February 1997): WP403-WP403.
Source
wos-lite
Published In
Biophysical Journal
Volume
72
Issue
2
Publish Date
1997
Start Page
WP403
End Page
WP403

Cloning and sequences of QPS3 of mitochondrial succinate-Q reductase.

Authors
Shenoy, SK; Yu, L; Yu, CA
MLA Citation
Shenoy, SK, Yu, L, and Yu, CA. "Cloning and sequences of QPS3 of mitochondrial succinate-Q reductase." FASEB JOURNAL 10.6 (April 30, 1996): 2837-2837.
Source
wos-lite
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
2837
End Page
2837

Cloning and sequencing of QPS3 of mitochondrial succinate -Q reductase

Bovine heart mitochondnal succinate-ubiquinone reductase (SQR),which catalyses electron transfer from succinate to ubiquinone, is composed of a soluble succinate dehydrogenase (SDK) and a membrane anchoring protein fraction (QPs). QPs forms Q-binding site(s) and serves as a membrane anchor for SDK, converting it to a TTFA-sensitive SQR. This fraction contains three protein subunits named QPsl(lSkDa), QPs2 (13kDa), and QPs3 (llkDa). QPsl has been cloned and sequenced and its Q-binding domain located. The protein sequences of QPs2 and QPs3 are not yet available. Recently, pure QPs3 protein was isolated and a partial N-terminal amino acid sequence (42 residues) was determined. This enabled us to obtain the cDNA encoding QPs3 from a bovine heart cDNA library using PCR techniques. The QPs3 cDNA is 1150 bp with an open reading frame of 474 bp which encodes a 103amino acid mature QPs3 and a 55-amino acid presequence. The molecular weight of QPs3, calculated from the deduced amino acid sequence, is 10989 daltons. QPs3 is a very hydrophobic protein, with three transmembrane helices revealed by a hydropathy plot of the sequence. The deduced sequence was confirmed by 5 additional peptide sequences obtained from pure QPs3.

Authors
Shenoy, SK; Yu, L; Yu, C-A
MLA Citation
Shenoy, SK, Yu, L, and Yu, C-A. "Cloning and sequencing of QPS3 of mitochondrial succinate -Q reductase." FASEB Journal 10.6 (1996): A1492-.
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
A1492

Identification of amino acid residues involved in structural and ubiquinone-binding functions of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides.

Previous studies established that subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides is involved in structural and ubiquinone-binding functions of the complex. To identify regions or amino acid residues responsible for these functions, deletion, insertion, and substitution mutations at various regions of subunit IV were generated and characterized. Mutational effects on the structural role of subunit IV are indicated by a delay in photosynthetic growth and by a decrease in the cytochrome bc1 complex activity in chromatophores upon detergent treatment. An effect on the ubiquinone-binding function of subunit IV is suggested by an increase in the apparent Km for 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol (Q2H2) of the complex. RSIV delta (2-5), in which residues 2-5 are deleted, had photosynthetic growth behavior, tolerance to detergent treatment, and an apparent Km for Q2H2 of its cytochrome bc1 complex similar to those of wild-type or complement cells, indicating that amino acid residues 2-5 are not essential for subunit IV function. RSIV delta (2-11), with residues 2-11 missing, showed a 24-h delay in photosynthetic growth and a 65% inactivation of the cytochrome bc1 complex upon dodecyl maltoside solubilization. However, its apparent Km for Q2H2 was the same as in wild-type cells, indicating that deletion of amino acid residues 6-11 results in loss of the structural but not the ubiquinone-binding function of subunit IV. RSIV delta (113-124), which has 13 amino acid residues deleted from the C terminus, had photosynthetic growth behavior, tolerance to detergent treatment, and ubiquinone-binding kinetics similar to those of wild-type or complement cells, indicating that residues 113-124 are not essential. Point mutants RSIV(W79L) and RSIV(W79F), in which tryptophan 79 was replaced with leucine or phenylalanine, showed a 24-h delay in photosynthetic growth, a decrease of 75% of the cytochrome bc1 complex activity in chromatophores upon detergent solubilization, and a 4-fold increase in the apparent Km for Q2H2, indicating that Trp-79 is essential for the structural and ubiquinone-binding functions of subunit IV.

Authors
Chen, YR; Shenoy, SK; Yu, CA; Yu, L
MLA Citation
Chen, YR, Shenoy, SK, Yu, CA, and Yu, L. "Identification of amino acid residues involved in structural and ubiquinone-binding functions of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides." J Biol Chem 270.19 (May 12, 1995): 11496-11501.
PMID
7744789
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
19
Publish Date
1995
Start Page
11496
End Page
11501
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