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Jeffrey L. Benovic - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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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.
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structure and function of the third intracellular loop of the 5 hydroxytryptamine2a receptor the third intracellular loop is α helical and binds purified Arrestins
Journal of Neurochemistry, 2008Co-Authors: Edward I Gelber, Jeffrey L. Benovic, Vsevolod V Gurevich, Wesley K Kroeze, David L Willins, John A Gray, Christine A Sinar, Edward Hyde, Bryan L RothAbstract:Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine2A (5-HT2A) receptor, a model G(alpha)q-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with alpha-helical and beta-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several Arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known Arrestins (beta-Arrestin, Arrestin-3, and visual Arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-Arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed Arrestins (beta-Arrestin or Arrestin-3) and 5-HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT2A receptor represents a structurally ordered domain composed of alpha-helical and beta-loop, -turn, and -sheet regions, (b) that this loop interacts with Arrestins in vitro, and is hence active, and (c) that Arrestins are colocalized with 5-HT2A receptors in vivo.
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haloperidol and clozapine differentially affect the expression of Arrestins receptor kinases and extracellular signal regulated kinase activation
Journal of Pharmacology and Experimental Therapeutics, 2008Co-Authors: Mohamed Rafiuddin Ahmed, Vsevolod V Gurevich, Jeffrey L. Benovic, Kevin N Dalby, Eugenia V GurevichAbstract:Dopamine and other G protein-coupled receptors (GPCRs) represent the major target of antipsychotic drugs. GPCRs undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by Arrestin binding. Arrestins and GRKs are major regulators of GPCR signaling. We elucidated changes in expression of two Arrestins and four GRKs following chronic (21 days) treatment with haloperidol (1 mg/kg i.p.) or clozapine (20 mg/kg i.p.) 2 or 24 h after the last injection in 11 brain regions. Haloperidol decreased GRK3 in ventrolateral caudate-putamen and transiently down-regulated GRK5 in globus pallidus and caudal caudate-putamen. Clozapine also caused a short-term suppression of the GRK5 expression in the caudal caudate-putamen and globus pallidus, but, unlike haloperidol, elevated GRK5 in the caudal caudate-putamen after 24 h. Unlike haloperidol, clozapine decreased Arrestin2 and GRK3 in hippocampus and GRK3 in globus pallidus but increased Arrestin2 in the core of nucleus accumbens and ventrolateral caudate-putamen and GRK2 in prefrontal cortex. Clozapine, but not haloperidol, induced long-term activation of extracellular signal-regulated kinase (ERK) 2 in ventrolateral caudate-putamen and transient in prefrontal cortex. The data demonstrate that haloperidol and clozapine differentially affect the expression of Arrestins and GRKs and ERK activity, which may play a role in determining their clinical profile.
Vsevolod V Gurevich - One of the best experts on this subject based on the ideXlab platform.
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plethora of functions packed into 45 kda Arrestins biological implications and possible therapeutic strategies
Cellular and Molecular Life Sciences, 2019Co-Authors: Vsevolod V Gurevich, Eugenia V GurevichAbstract:Mammalian Arrestins are a family of four highly homologous relatively small ~ 45 kDa proteins with surprisingly diverse functions. The most striking feature is that each of the two non-visual subtypes can bind hundreds of diverse G protein-coupled receptors (GPCRs) and dozens of non-receptor partners. Through these interactions, Arrestins regulate the G protein-dependent signaling by the desensitization mechanisms as well as control numerous signaling pathways in the G protein-dependent or independent manner via scaffolding. Some partners prefer receptor-bound Arrestins, some bind better to the free Arrestins in the cytoplasm, whereas several show no apparent preference for either conformation. Thus, Arrestins are a perfect example of a multi-functional signaling regulator. The result of this multi-functionality is that reduction (by knockdown) or elimination (by knockout) of any of these two non-visual Arrestins can affect so many pathways that the results are hard to interpret. The other difficulty is that the non-visual subtypes can in many cases compensate for each other, which explains relatively mild phenotypes of single knockouts, whereas double knockout is lethal in vivo, although cultured cells lacking both Arrestins are viable. Thus, deciphering the role of Arrestins in cell biology requires the identification of specific signaling function(s) of Arrestins involved in a particular phenotype. This endeavor should be greatly assisted by identification of structural elements of the Arrestin molecule critical for individual functions and by the creation of mutants where only one function is affected. Reintroduction of these biased mutants, or introduction of monofunctional stand-alone Arrestin elements, which have been identified in some cases, into double Arrestin-2/3 knockout cultured cells, is the most straightforward way to study Arrestin functions. This is a laborious and technically challenging task, but the upside is that specific function of Arrestins, their timing, subcellular specificity, and relations to one another could be investigated with precision.
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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.
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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.
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role of receptor attached phosphates in binding of visual and non visual Arrestins to g protein coupled receptors
Journal of Biological Chemistry, 2012Co-Authors: Luis E Gimenez, Eugenia V Gurevich, Sergey A Vishnivetskiy, Seunghyi Kook, Rafiuddin M Ahmed, Vsevolod V GurevichAbstract:Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual Arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual Arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in Arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of Arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in Arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual Arrestins in which the K2A mutations had little effect on net BRETmax values for the M2 muscarinic acetylcholine (M2R), β2-adrenergic (β2AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual Arrestins normally, whereas phosphorylation-deficient β2AR mutants bound Arrestins at 20–50% of the level of wild type β2AR. Thus, the contribution of receptor-attached phosphates to Arrestin binding varies depending on the receptor-Arrestin pair. Although Arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of Arrestin-2 and -3 is much greater in the case of β2AR than M2R and D2 dopamine receptor.
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nonvisual Arrestins function as simple scaffolds assembling the mkk4 jnk3α2 signaling complex
Biochemistry, 2011Co-Authors: Xuanzhi Zhan, Kevin N Dalby, Tamer S Kaoud, Vsevolod V GurevichAbstract:Arrestins are a small family of proteins with four mammalian members that play key roles in the regulation of multiple GPCR-dependent and -independent signaling pathways. Although Arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated and even the direct binding of Arrestins with MAP kinases were never demonstrated. Here using purified proteins we show that both non-visual Arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of Arrestin-3 for these kinases is higher than that of Arrestin-2. Reconstitution of the MKK4-JNK3α2 signaling module from pure proteins in the presence of different Arrestin-3 concentrations showed that Arrestin-3 acts as a “true” scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both JNK3α2 phosphorylation by MKK4 and JNK3α2 activity towards its substrate ATF2 increase at low and then decrease at high Arrestin-3 levels, yielding bell-shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of Arrestin-3 action on the MKK4-JNK3α2 signaling module.
Nigel W Bunnett - One of the best experts on this subject based on the ideXlab platform.
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endothelin converting enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β Arrestins
Journal of Cell Biology, 2007Co-Authors: Benjamin E Padilla, Graeme S Cottrell, Dirk Roosterman, Stella Pikios, Laurent Muller, Martin Steinhoff, Nigel W BunnettAbstract:Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-Arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-Arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-Arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-Arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-Arrestin complex, freeing internalized receptors from β-Arrestins and promoting recycling and resensitization.
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endothelin converting enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and beta Arrestins
Journal of Cell Biology, 2007Co-Authors: Benjamin E Padilla, Graeme S Cottrell, Dirk Roosterman, Stella Pikios, Laurent Muller, Martin Steinhoff, Nigel W BunnettAbstract:Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-Arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-Arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-Arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-Arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-Arrestin complex, freeing internalized receptors from beta-Arrestins and promoting recycling and resensitization.
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trafficking of proteinase activated receptor 2 and β Arrestin 1 tagged with green fluorescent protein β Arrestin dependent endocytosis of a proteinase receptor
Journal of Biological Chemistry, 1999Co-Authors: Olivier Dery, Helen Wong, Eileen F Grady, Mark Thoma, Nigel W BunnettAbstract:Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. beta-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of beta-Arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of beta-Arrestins have not been examined. We studied trafficking of PAR2 and beta-Arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of beta-Arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of beta-Arrestin1 and PAR2 into the same early endosomes; (c) redistribution of beta-Arrestin1 to the cytosol concurrent with PAR2 translocation to lysosomes; and (d) mobilization of PAR2 from the Golgi apparatus to the plasma membrane. Overexpression of a C-terminal fragment of beta-Arrestin-319-418, which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PAR2. Our results show that beta-Arrestins mediate endocytosis of PAR2 and support a role for beta-Arrestins in uncoupling of PARs.
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substance p induced trafficking of β Arrestins the role of β Arrestins in endocytosis of the neurokinin 1 receptor
Journal of Biological Chemistry, 1999Co-Authors: Karen Mcconalogue, Olivier Dery, Michelle Lovett, Helen Wong, John H Walsh, Eileen F Grady, Nigel W BunnettAbstract:Abstract Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and β-Arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of β-Arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or β-Arrestin1. SP induced (a) striking and rapid ( 60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and β-Arrestin1 in the cytosol (4–6 h). SP stimulated a similar redistribution of immunoreactive β-Arrestin1 and β-Arrestin2. In contrast, SP did not affect Gαq/11distribution, which remained at the plasma membrane. Expression of the dominant negative β-Arrestin319–418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of β-Arrestins to the plasma membrane, where they participate in NK1R endocytosis. β-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and β-Arrestins return to the cytosol.
Sudha K Shenoy - One of the best experts on this subject based on the ideXlab platform.
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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.
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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.
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Arrestins and protein ubiquitination.
Progress in Molecular Biology and Translational Science, 2013Co-Authors: Reddy Peera Kommaddi, Sudha K ShenoyAbstract: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.
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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, Arun K Shukla, Seungkirl Ahn, Aalok S. Modi, Kunhong Xiao, Magali Berthouze, Keith D. Wilkinson, William E. MillerAbstract: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.
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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 LichtargeAbstract: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.
Stephen S. G. Ferguson - One of the best experts on this subject based on the ideXlab platform.
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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.
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cellular trafficking of g protein coupled receptor beta Arrestin endocytic complexes
Journal of Biological Chemistry, 1999Co-Authors: Jie Zhang, 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.
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cellular trafficking of g protein coupled receptor beta Arrestin endocytic complexes
Journal of Biological Chemistry, 1999Co-Authors: Jie Zhang, 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.
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a β Arrestin green fluorescent protein biosensor for detecting g protein coupled receptor activation
Journal of Biological Chemistry, 1997Co-Authors: Larry S Barak, Jie Zhang, Stephen S. G. Ferguson, Marc G CaronAbstract:G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a β-Arrestin2/green fluorescent protein conjugate (βarr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-G protein-coupled receptor kinase or GPCR-Arrestin interactions. Confocal microscopy demonstrates the translocation of βarr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the β-Arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with β-Arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active β-Arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of β-Arrestin activity and GPCR signal transduction in living cells. The use of βarr2-GFP as a biosensor to recognize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.