The Experts below are selected from a list of 16410 Experts worldwide ranked by ideXlab platform
Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
-
sortase ligation enables homogeneous gpcr phosphorylation to reveal diversity in β arrestin coupling
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Robert J Lefkowitz, Aashish Manglik, Andrew C Kruse, Dean P Staus, Laura M Wingler, Minjung Choi, Biswaranjan PaniAbstract:The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and β-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer’s mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in β-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and β-Arrestins with GPCRs’ transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the β 2 -adrenergic receptor (β 2 AR) allows stabilization of a high-affinity receptor active state by β-arrestin1, permitting us to define elements in the β 2 AR and β-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the β 2 AR, the muscarinic acetylcholine receptor 2 and the μ-opioid receptor reveals that the ability of β-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of β-arrestin1 correlates with its ability to attenuate, or “desensitize,” G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor–transducer interactions.
-
β-Arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Sudha K Shenoy, William E. Miller, Arun K Shukla, Seungkirl Ahn, Aalok S. Modi, Kunhong Xiao, Magali Berthouze, Keith D. Wilkinson, Robert J LefkowitzAbstract: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.
-
β arrestin scaffolding of phosphatidylinositol 4 phosphate 5 kinase iα promotes agonist stimulated sequestration of the β2 adrenergic receptor
Journal of Biological Chemistry, 2008Co-Authors: Christopher D. Nelson, Erin J Whalen, Jeffery J Kovacs, Kelly Nobles, Robert J LefkowitzAbstract:Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-Arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-Arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-Arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-Arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-Arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.
-
β Arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration
Circulation Research, 2008Co-Authors: Jihee Kim, Robert J Lefkowitz, Lisheng Zhang, Karsten Peppel, David A Zidar, Leigh Brian, Scott M Dewire, Sabrina T Exum, Neil J FreedmanAbstract:Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.
-
β arrestin2 mediated inotropic effects of the angiotensin ii type 1a receptor in isolated cardiac myocytes
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Keshava Rajagopal, Richard T. Premont, Erin J Whalen, Jonathan D Violin, Jonathan A Stiber, Paul B Rosenberg, Thomas M Coffman, Howard A Rockman, Robert J LefkowitzAbstract:The G protein-coupled receptor kinases (GRKs) and β-Arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/β-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/β-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT1AR) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the “biased” AT1AR agonist [Sar1, Ile4, Ile8]-angiotensin II (SII), which is unable to stimulate Gαq-mediated signaling, but which has previously been shown to promote β-arrestin interaction with the AT1AR. Cardiomyocytes from WT, but not AT1AR-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from β-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2+/−) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent Gαq/PKC- and GRK6/β-arrestin2-dependent mechanisms by which stimulation of the AT1AR can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.
Vsevolod V Gurevich - One of the best experts on this subject based on the ideXlab platform.
-
Exploring GPCR‐arrestin interfaces with genetically encoded crosslinkers
EMBO reports, 2020Co-Authors: Thore Böttke, Vsevolod V Gurevich, Stefan Ernicke, Robert Serfling, Christian Ihling, Edyta Burda, Andrea Sinz, Irene CoinAbstract:β-Arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that β-Arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of Arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with Arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on Arrestins, whereas the two β-Arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.
-
heterologous phosphorylation induced formation of a stability lock permits regulation of inactive receptors by β Arrestins
Journal of Biological Chemistry, 2017Co-Authors: Andras Toth, Vsevolod V Gurevich, Susanne Prokop, Pal Gyombolai, Peter Varnai, Andras Balla, Laszlo HunyadyAbstract:-Arrestins are key regulators and signal transducers of G protein– coupled receptors (GPCRs). The interaction between receptors and -Arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether -Arrestins are able to bind second messenger kinase–phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of -arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes -arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and -Arrestins, formed by phosphorylated serine–threonine clusters in the receptor’s C terminus and two conserved phosphate-binding lysines in the -arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters -arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor–-arrestin interaction, but also governs the structural rearrangements within -Arrestins. Furthermore, we found that -arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of -Arrestins and reveal their novel role in receptor cross-talk. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
-
Arrestin-3: The Structural Basis of Lower Receptor Selectivity
The Structural Basis of Arrestin Functions, 2017Co-Authors: Benjamin W. Spiller, Xuanzhi Zhan, Vsevolod V GurevichAbstract:Arrestin-3 (β-arrestin2) is the second member of the non-visual arrestin subfamily cloned. Together with arrestin-2 (β-arrestin1), it is responsible for arrestin function outside the visual system. Arrestins 2 and 3 are multi-functional proteins, originally identified as important for termination of signaling through G protein-coupled receptors (GPCRs), but now recognized as versatile regulators of signaling and trafficking of most GPCRs, as well as signaling switches controlling the balance between G protein-dependent and -independent GPCR signaling. Arrestin-3 is notably the least selective arrestin family member in that it binds a wide variety of GPCRs and that binding is less dependent on receptor activation and phosphorylation than for other arrestin subtypes. A recently determined arrestin-3 structure reveals that the basal conformation of arrestin-3 is similar to other Arrestins, and that similar molecular interactions stabilize the basal state in all Arrestins. A disruption of a structural element in the C-domain of arrestin-3 has been implicated as the cause of the reduced selectivity of arrestin-3. Here we compare the basal arrestin-3 structure with the recently determined structure of an activated receptor-bound arrestin-1.
-
GPCR Footprint on Arrestins and Manipulation of Receptor Specificity
The Structural Basis of Arrestin Functions, 2017Co-Authors: Sergey A Vishnivetskiy, Candice S. Klug, Wayne L. Hubbell, Vsevolod V GurevichAbstract:A wide variety of methods were used to identify where exactly do GPCRs bind Arrestins. Experiments implicated large part of the concave sides of the two arrestin domains and identified surprisingly few residues on this extensive surface that determine which receptor does each arrestin prefer. Manipulation of these “receptor-discriminator” residues in one of the non-visual subtypes, arrestin-3, yielded non-visual Arrestins with greatly enhanced receptor specificity. Targeting particular receptors with arrestin mutants with special functional characteristics, such as the ability to bind unphosphorylated GPCRs, activate or fail to activate select pathways of arrestin-mediated signaling opens the prospect of creating “designer” Arrestins to direct the signaling from GPCRs of our choice to the pathways we want.
-
identification of receptor binding induced conformational changes in non visual Arrestins
Journal of Biological Chemistry, 2014Co-Authors: Ya Zhuo, Vsevolod V Gurevich, Xuanzhi Zhan, Sergey A Vishnivetskiy, Candice S. KlugAbstract:The non-visual Arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual Arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual Arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual Arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual Arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual Arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.
Jeffrey L. Benovic - One of the best experts on this subject based on the ideXlab platform.
-
Structure of an arrestin2-clathrin complex reveals a novel clathrin binding domain that modulates receptor trafficking.
2018Co-Authors: Dong Soo Kang, Ronald C. Kern, Manojkumar Puthenveedu, Mark Von Zastrow, John C. Williams, Jeffrey L. BenovicAbstract:Non-visual Arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin beta-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)(2)GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.
-
β-Arrestins and G protein-coupled receptor trafficking.
Methods in Enzymology, 2013Co-Authors: Dong Soo Kang, Xufan Tian, Jeffrey L. BenovicAbstract:Arrestins are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. There are four mammalian members of the arrestin family, two visual and two nonvisual. The visual Arrestins (arrestin-1 and arrestin-4) are localized in rod and cone cells, respectively, and function to quench phototransduction by inhibiting receptor/G protein coupling. The nonvisual Arrestins (β-arrestin1 and β-arrestin2, a.k.a. arrestin-2 and arrestin-3) are ubiquitously expressed and function to inhibit GPCR/G protein coupling and promote GPCR trafficking and arrestin-mediated signaling. Arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of β-Arrestins with clathrin, adaptor protein 2, and phosphoinositides such as PIP2/PIP3. These interactions are facilitated by a conformational change in β-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this chapter, we provide an overview of the reagents and techniques used to study β-arrestin-mediated receptor trafficking.
-
β-Arrestins and G protein-coupled receptor trafficking.
Handbook of experimental pharmacology, 2013Co-Authors: Xufan Tian, Dong Soo Kang, Jeffrey L. BenovicAbstract:Nonvisual Arrestins (β-arrestin-1 and β-arrestin-2) are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. β-Arrestins are ubiquitously expressed and function to inhibit GPCR/G protein coupling, a process called desensitization, and promote GPCR trafficking and arrestin-mediated signaling. β-arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of β-Arrestins with clathrin, adaptor protein 2 (AP2), and phosphoinositides. These interactions are facilitated by a conformational change in β-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this review, we provide an overview of the key interactions involved in β-arrestin-mediated trafficking of GPCRs.
-
Differential expression of Arrestins is a predictor of breast cancer progression and survival
Breast Cancer Research and Treatment, 2011Co-Authors: Allison M. Michal, Amy R. Peck, Thai H. Tran, Chengbao Liu, David L. Rimm, Hallgeir Rui, Jeffrey L. BenovicAbstract:Emerging evidence has implicated G protein-coupled receptors, such as CXCR4 and PAR2, in breast cancer progression and the development of metastatic breast cancer. However, the role of proteins that regulate the function of these receptors, such as Arrestins, in breast cancer has yet to be determined. Examination of the expression of the two nonvisual Arrestins, arrestin2 and 3, in various breast cancer cell lines revealed comparable expression of arrestin3 in basal and luminal lines while arrestin2 expression was much higher in the luminal lines compared to the more aggressive basal lines. Analysis of normal human breast tissue revealed that arrestin2 and 3 were expressed in both luminal and myoepithelial cells of mammary epithelia with arrestin2 highest in myoepithelial cells and arrestin3 comparable in both cell types. Quantitative immunofluorescence-based examination of primary breast tumors revealed that arrestin2 expression significantly decreased with cancer progression from ductal carcinoma in situ to invasive carcinoma and further to lymph node metastasis ( P
-
Non-visual Arrestins Are Constitutively Associated with the Centrosome and Regulate Centrosome Function
Journal of Biological Chemistry, 2010Co-Authors: Haripriya Shankar, Dong Soo Kang, Vsevolod V Gurevich, Allison M. Michal, Ronald C. Kern, Jeffrey L. BenovicAbstract:In addition to regulating receptor activity, non-visual Arrestins function as scaffolds for numerous intracellular signaling cascades and as regulators of gene transcription. Here we report that the two non-visual Arrestins, arrestin2 and arrestin3, localize to the centrosome, a key organelle involved in microtubule nucleation and bipolar mitotic spindle assembly. Both Arrestins co-localized with the centrosomal marker γ-tubulin during interphase and mitosis and were found in purified centrosome preparations. In vitro binding assays demonstrated that both Arrestins directly interact with γ-tubulin. Knockdown of either arrestin by RNA interference resulted in multinucleation, centrosome amplification, and mitotic defects, although only the loss of arrestin2 triggered aberrant microtubule nucleation. Importantly, overexpression of wild type arrestin rescued the multinucleation phenotype and restored normal centrosome number in arrestin siRNA-transfected cells. Moreover, overexpression of arrestin2 or -3 rescued the multinucleation defect observed in MDA-MB-231 breast cancer cells. Taken together, our data reveal that non-visual Arrestins are novel centrosomal components and regulate normal centrosome function.
Sudha K Shenoy - One of the best experts on this subject based on the ideXlab platform.
-
regulation of inflammation by β Arrestins not just receptor tales
Cellular Signalling, 2018Co-Authors: Neil J Freedman, Sudha K ShenoyAbstract: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.
-
ubiquitin related roles of β Arrestins in endocytic trafficking and signal transduction
Journal of Cellular Physiology, 2016Co-Authors: Pierreyves Jeancharles, Vishwaesh Rajiv, Sudha K ShenoyAbstract: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.
-
β-Arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Sudha K Shenoy, William E. Miller, Arun K Shukla, Seungkirl Ahn, Aalok S. Modi, Kunhong Xiao, Magali Berthouze, Keith D. Wilkinson, Robert J LefkowitzAbstract: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.
-
β arrestin dependent g protein independent erk1 2 activation by the β2 adrenergic receptor
Journal of Biological Chemistry, 2006Co-Authors: Sudha K Shenoy, Richard T. Premont, Kunhong Xiao, Matthew T. Drake, Christopher D. Nelson, Daniel A. Houtz, Srinivasan Madabushi, Eric Reiter, Olivier Lichtarge, Robert J LefkowitzAbstract:Abstract Physiological effects of β adrenergic receptor (β2AR) stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein β-Arrestins mediate β2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the β2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on Gs-Gi/protein kinase A (PKA) or β-Arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (Gi inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous β-Arrestins by siRNA. β-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 β-arrestin1 or -2 by small interfering RNA. In Gs knock-out mouse embryonic fibroblasts, wild-type β2AR recruited β-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel β2AR mutant (β2ART68F,Y132G,Y219A or β2ARTYY), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit β-Arrestins, undergo β-arrestin-dependent internalization, and activate β-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and β-arrestin recruitment, led to the formation of stable receptor-β-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gβγ 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 β2AR can signal to ERK via a GRK5/6-β-arrestin-dependent pathway, which is independent of G protein coupling.
-
β arrestin dependent g protein independent erk1 2 activation by the β2 adrenergic receptor
Journal of Biological Chemistry, 2006Co-Authors: Sudha K Shenoy, Richard T. Premont, Kunhong Xiao, Matthew T. Drake, Christopher D. Nelson, Daniel A. Houtz, Srinivasan Madabushi, Eric Reiter, Olivier Lichtarge, Robert J LefkowitzAbstract: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.
Stephen S. G. Ferguson - One of the best experts on this subject based on the ideXlab platform.
-
The fate of the internalized apelin receptor is determined by different isoforms of apelin mediating differential interaction with β-arrestin
Biochemical and biophysical research communications, 2010Co-Authors: Dennis K. Lee, Stephen S. G. Ferguson, Susan R. George, Brian F. O'dowdAbstract:Internalization of the apelin receptor by apelin-13 is characterized by dissociation from beta-Arrestins and rapid recycling to the cell surface. Paradoxically, the apelin receptor internalized by apelin-36 was sequestered intracellularly. The specific pathways involved in apelin receptor trafficking were resolved using beta-arrestin1 and constitutively active and dominant negative Rab proteins following activation by apelin-13 or apelin-36. beta-Arrestin1 dissociated from the apelin-13-internalized receptor while the apelin-36-internalized receptor was trafficked with beta-arrestin1 to intracellular compartments. The apelin-13-internalized receptor was rapidly recycled to the cell surface through a Rab4-dependent mechanism while Rab7 targeted the receptor to lysosomes. The internalized receptor co-expressed with dominant negative Rab4 were trafficked to lysosomes. These observations revealed a novel ligand-dependent targeting of the apelin receptor to beta-arrestin-associated and -dissociated trafficking pathways and a role for different Rab proteins to direct these pathways.
-
agonist stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells agonist stimulated endocytosis is β arrestin1 isoform specific
Molecular Pharmacology, 2001Co-Authors: Lianne B Dale, Pieter H. Anborgh, Moshmi Bhattacharya, Jennifer L Seachrist, Stephen S. G. FergusonAbstract:Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and γ-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether β-Arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by β-arrestin1 in HEK 293 cells. However, even though β-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither β-arrestin1 nor β-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, β-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, β2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with β-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the β-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.
-
Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes.
The Journal of biological chemistry, 1999Co-Authors: Jie Zhang, Marc G Caron, Larry S Barak, Stephane A Laporte, Pieter H. Anborgh, Stephen S. G. FergusonAbstract:beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-Arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-Arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-Arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-Arrestins.
-
cellular trafficking of g protein coupled receptor beta arrestin endocytic complexes
Journal of Biological Chemistry, 1999Co-Authors: Jie Zhang, Marc G Caron, Larry S Barak, Stephane A Laporte, Pieter H. Anborgh, Stephen S. G. FergusonAbstract:beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-Arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-Arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-Arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-Arrestins.
-
cellular trafficking of g protein coupled receptor beta arrestin endocytic complexes
Journal of Biological Chemistry, 1999Co-Authors: Jie Zhang, Marc G Caron, Larry S Barak, Stephane A Laporte, Pieter H. Anborgh, Stephen S. G. FergusonAbstract:Abstract β-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, β-Arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of β-arrestin2 in response to activation of several distinct GPCRs including the β2-adrenergic receptor (β2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to β2AR activation, β-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for β-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed β-Arrestins are confined to the periphery of cells and do not traffic along with activated β2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of β-Arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the β2AR carboxyl-terminal tail results in a β-arrestin membrane localization pattern similar to that observed in response to β2AR activation. In contrast, the corresponding chimeric β2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate β-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of β-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/βarrestin complexes and cellular distribution of β-Arrestins.