Protein Β-arrestin Research Articles

Novel Structural Insights into GPCR-β-Arrestin Interaction and Signaling.

G protein-coupled receptors (GPCRs) are major signal recognition and transmission units in the plasma membrane. The interaction of activated and phosphorylated GPCRs with the multifunctional adaptor proteins β-arrestins (βarrs) is crucial for regulation of their signaling and functional outcomes. Over the past few years, a range of structural, biochemical, and cellular studies have revealed novel insights into GPCR-βarr interaction and signaling. Some of these findings have come as a surprise and therefore have the potential to significantly refine the conceptual framework of the GPCR-βarr system. Here we discuss these recent advances with particular emphasis on biphasic GPCR-βarr interaction, the formation of GPCR-G-protein-βarr supercomplexes, and receptor-specific conformational signatures in βarrs. We also underline the emerging research areas that are likely to be at the center stage of investigations in the coming years.

Ligand-Specific Restriction of Extracellular Conformational Dynamics Constrains Signaling of the M2 Muscarinic Receptor.

G protein-coupled receptors transmit extracellular signals across cell membranes via different G protein classes and β-arrestins. Some pathways may be therapeutically beneficial, whereas others may be detrimental under certain pathophysiological conditions. For many GPCRs, biased agonists are available, which preferentially signal through one pathway or a subset of pathways, and harnessing biased agonism could be a potential novel therapeutic strategy. However, the incomplete mechanistic understanding of biased agonism hampers rational design of biased ligands. Using the muscarinic M2 receptor as a model system, we have analyzed the relationship between ligand-dependent conformational changes as revealed in all-atom MD simulations and the activation of specific G proteins. We find that the extent of closure of the extracellular, allosteric binding site interferes with the activation of certain G proteins. Our data allow the rational design of Gi-biased agonists at the M2 receptor and delineate a simple principle which may be translated to other GPRCs.

Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that is a major therapeutic target for the treatment of type 2 diabetes. Activation of this receptor promotes insulin secretion and blood glucose regulation. The GLP-1R can initiate signaling through several intracellular pathways upon activation by GLP-1. GLP-1R ligands that preferentially stimulate subsets among the natural signaling pathways (“biased agonists”) could be useful as tools for elucidating the consequences of specific pathways and might engender therapeutic agents with tailored effects. Using HEK-293 cells recombinantly expressing human GLP-1R, we have previously reported that backbone modification of GLP-1, via replacement of selected α-amino acid residues with β-amino acid residues, generates GLP-1 analogues with distinctive preferences for promoting G protein activation versus β-arrestin recruitment. Here, we have explored the influence of cell background across these two parameters and expanded our analysis to include affinity and other key signaling pathways (intracellular calcium mobilization and ERK phosphorylation) using recombinant human GLP-1R expressed in a CHO cell background, which has been used extensively to demonstrate biased agonism of GLP-1R ligands. The new data indicate that α/β-peptide analogues of GLP-1 exhibit a range of distinct bias profiles relative to GLP-1 and that broad assessment of signaling endpoints is required to reveal the spectrum of behavior of modified peptides. These results support the view that backbone modification via α→β amino acid replacement can enable rapid discovery of peptide hormone analogues that display substantial signal bias at a cognate GPCR.

Arresting the Development of Addiction: The Role of β-Arrestin 2 in Drug Abuse.

The protein β-arrestin (βarr) 2 directly interacts with receptors and signaling pathways that mediate the behavioral effects of drugs of abuse, making it a prime candidate for therapeutic interventions. βarr2 drives desensitization and internalization of G protein-coupled receptors, including dopamine, opioid, and cannabinoid receptors, and it can also trigger G protein-independent intracellular signaling. βarr2 mediates several drug-induced behaviors, but the relationship is complex and dependent on the type of behavior (e.g., psychomotor versus reward), the class of drug (e.g., psychostimulant versus opioid), and the circuit being interrogated (e.g., brain region, cell type, and specific receptor ligand). Here we discuss the current state of research concerning the contribution of βarr2 to the psychomotor and rewarding effects of addictive drugs. Next we identify key knowledge gaps and suggest new tools and approaches needed to further elucidate the neuroanatomical substrates and neurobiological mechanisms to explain how βarr2 modulates behavioral responses to drugs of abuse, as well as its potential as a therapeutic target.

Assays of adrenal GPCR signaling and regulation: Measuring adrenal β-arrestin activity in vivo through plasma membrane recruitment.

Production and secretion of catecholamines and aldosterone from the adrenal gland are regulated by various adrenal G protein-coupled receptors (GPCRs), including α2-adrenergic (auto)receptors (ARs) and angiotensin receptors. These hormones, in turn, regulate cardiac function and play important pathophysiological roles in heart failure (HF). Recent studies have identified the GPCR adapter proteins β-arrestins, specifically β-arrestin1, as an important mediator of the GPCR-dependent production of these hormones in the adrenal gland. Upon GPCR agonist activation, β-arrestins translocate from the cytoplasm to the plasma membrane to interact with the phosphorylated and active receptor and mediate its G protein-independent signaling. Here, we describe a simple and fast Western blotting-based technique for measuring adrenal β-arrestin plasma membrane recruitment in vivo, as an (indirect) index of its activity toward various agonist-activated adrenal GPCRs in vivo.

Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines.

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with β-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CB1R, which we hypothesized could attenuate the action of β-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, β-arrestin-mediated internalization of the CB1R.

β-Arrestin mediates the Frank–Starling mechanism of cardiac contractility

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein β-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking β-arrestin 1, β-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a β-arrestin-biased AT1R ligand to selectively activate β-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in β-arrestin 1, β-arrestin 2, and AT1R knockout mice. Our data reveal β-arrestin 1, β-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with β-arrestin-biased AT1R ligands.

Identification of the First Marine-Derived Opioid Receptor "Balanced" Agonist with a Signaling Profile That Resembles the Endorphins.

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are "biased" agonists at the μ opioid receptor (OR), wherein they engage G protein signaling but poorly engage β-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and β-arrestin pathways as "balanced" agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)-aaptamine (12) with activity at the δ-OR (EC50: 5.1, 4.1, 2.3 μM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the μ-OR (EC50: 6.3, 4.2 μM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and β-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.

β-Arrestin-Biased Agonists of the GLP-1 Receptor from β-Amino Acid Residue Incorporation into GLP-1 Analogues.

Activation of a G protein-coupled receptor (GPCR) causes recruitment of multiple intracellular proteins, each of which can activate distinct signaling pathways. This complexity has engendered interest in agonists that preferentially stimulate subsets among the natural signaling pathways ("biased agonists"). We have examined analogues of glucagon-like peptide-1 (GLP-1) containing β-amino acid residues in place of native α residues at selected sites and found that some analogues differ from GLP-1 in terms of their relative abilities to promote G protein activation (as monitored via cAMP production) versus β-arrestin recruitment (as monitored via BRET assays). The α → β replacements generally cause modest declines in stimulation of cAMP production and β-arrestin recruitment, but for some replacement sets cAMP production is more strongly affected than is β-arrestin recruitment. The central portion of GLP-1 appears to be critical for achieving bias toward β-arrestin recruitment. These results suggest that backbone modification via α → β residue replacement may be a versatile source of agonists with biased GLP-1R activation profiles.

Design and Discovery of Functionally Selective Serotonin 2C (5-HT2C) Receptor Agonists.

On the basis of the structural similarity of our previous 5-HT2C agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT2A, 5-HT2B, and 5-HT2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment. The most functionally selective compound (+)-7e produced weak β-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT2C agonists possessing weak β-arrestin recruitment can produce distinct receptor desensitization properties.

β-arrestin1 at the cross-road of endothelin-1 signaling in cancer

The advent of targeted therapeutics in human cancer has begun to find novel druggable targets and, in this context, the endothelin-1 receptor (ET-1R), namely ETA receptor (ETAR) and ETB receptor, among the GPCR family represents a class of highly druggable molecules in cancer. ET-1R are aberrantly expressed in human malignancies, potentially representing prognostic factors. Their activation by ligand stimulation initiate signaling cascades activating different downstream effectors, allowing precise control over multiple signaling pathways. ET-1R regulates cell proliferation, survival, motility, cytoskeletal changes, angiogenesis, metastasis as well as drug resistance. The molecular events underlying these responses are the activation of transcriptional factors and coactivators, and downstream genes, acting as key players in tumor growth and progression. ET-1R represent crucial cancer targets that have been exploited for ET-1R therapeutics. Importantly, efforts to explore new information of ETAR in cancer have uncovered that their functions are crucially regulated by multifunctional scaffold protein β-arrestins (β-arrs) which orchestrate the multidimensionality of ETAR signaling into highly regulated and distinct signaling complexes, a property that is highly advantageous for tumor signaling. Moreover, the role of β-arr1 in ET-1 signaling in cancer highlights why the pleiotropic effects of ET-1 and its dynamic signaling are more complex than previously recognized. In order to improve therapeutic strategies that interfere with the widespread effects of ET-1R, it is important to consider antagonists able to turn the receptors “off” selectively controlling β-arr1-dependent signaling, highlighting the possibility that targeting ETAR/β-arr1 may display a large therapeutic window in cancer.

Abstract 2901: Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression

Abstract In epithelial ovarian cancer (EOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) axis has been reported to be involved in invasiveness, epithelial-to-mesenchymal transition, chemoresistance and metastatic dissemination and is thereby associated with poor prognosis. In these cells, ET-1 is able to induce the nuclear translocation and stability of hypoxia-inducible factor-1α (HIF-1α) the principal transcriptional factor that allows cellular adaptation to hypoxia. The importance of co-factors that trigger an epigenetic regulation of HIF-1α within tumor is not well understood. Here we elucidate that ET-1/ETAR-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signaling. In EOC cells, ET-1 induced vascular-endothelial growth factor (VEGF), the pro-agiogenic target downstream of HIF-1α through HIF-1α nuclear accumulation. Activation of ET-1/ETAR axis, by mimicking hypoxia, promotes the nuclear association between β-arr1 and HIF-1α on the hypoxia responsive elements (HRE) binding sites. By ChIP assay, nuclear β-arr1 was able to interact with HIF-1α, by promoting the recruitment of p300 acetyltransferase. The formation of this functional complex resulted in enhanced histone acetylation, and gene transcription of HIF-1α downstream targets, such as ET-1 and VEGF, required for cell invasion. The induced release of ET-1 and VEGF by EOC cells, promoted angiogenic effects in endothelial cells, as evaluated by cell migration and tube-like structure formation assays. Interestingly ETAR/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit required for invasiveness and angiogenic responses. These effects were abrogated by the silencing of β-arr1 or HIF-1α or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. In a murine orthotropic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation, suggesting that ET-1, together with VEGF, represents one of such factors that mediates tumor-endothelial cell communications, favoring a permissive environment for metastatic spread. Collectively these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ETAR pathway, mediating epigenetic modifications, to an extent comparable with hypoxia, directly involved in metastatic progression. Targeting ET-1 axis by macitentan may represent a new opportunity to target the orchestration mediated by nuclear β-arr1/HIF-1α complex in EOC, demonstrating that this approved small molecule interfering with stromal compartment, expressing ETBR, and tumor cells, expressing mainly ETAR, can be used in clinical setting for improved therapeutics in EOC. Citation Format: Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Valentina Caprara, Rosanna Sestito, Valeriana Di Castro, Anna Bagnato. Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2901.

Highlights of This Issue

Mastermind-like 3 (MAML3) is a known co-activator of NOTCH signaling. Heynen and colleagues find an additional and unexpected role for MAML3 in retinoic acid signaling in neuroblastoma, which is used clinically to induce differentiation in this cancer. Elevated expression of MAML3 leads to loss of activation of a subset of retinoic acid receptor target genes and as a consequence blocks retinoic acid-induced differentiation. MAML3 also has retinoic acid independent functions in neuroblastoma through upregulation of IGF2, which activates AKT signaling downstream of IGF1R. These data shed light on why MAML3 is mutated in multiple cancers.Here, the functional and molecular consequences of cytotoxic chemotherapy treatment on pancreatic cancer-associated fibroblasts (CAFs) were investigated. Chemotherapy treated CAFs enhanced tumor cell viability, migration and invasion in vitro as well as tumor growth in vivo compared to treatment naïve CAFs. A pro-inflammatory gene expression signature mediated by stress-associated MAPK was identified in chemotherapy treated CAFs. Stress-associated MAPK inhibitors decreased expression of inflammatory mediators and reduced tumor cell viability, migration, and invasion in vitro as well as tumor growth in vivo. These results describe a novel molecular response to chemotherapy in pancreatic CAFs and represent a future therapeutic target.Increased signaling from growth-factor receptors and stress-related kinases plays a role in endocrine resistance, a major challenge in the treatment of estrogen receptor-positive breast cancer (BC) patients. An integrative in silico transcriptomic/cistromic approach implicated the AP-1 transcription complex as a signaling node in endocrine resistance. When AP-1 activity was inhibited by an inducible genetic approach in BC cells grown in vitro and in vivo, the blockade enhanced sensitivity to endocrine therapy, delayed resistance onset, and overcame the growth of tamoxifen-resistant tumors. These data demonstrate AP-1 as a driver of endocrine resistance and offer new perspectives for innovative treatment and biomarker research.The article by Kallifatidis and colleagues defines the heterotypic Src-mediated transactivation of the epidermal growth factor receptor (EGFR) by the atypical chemokine receptor, CXCR7, in prostate cancer. The highlight of the discovery is that the scaffold protein β-Arrestin 2 was found to regulate this transactivation and reduce the phosphorylation of EGFR at Tyrosyl 1110, one of the seven cytoplasmic phosphorylation domains of EGFR. Further, down-regulation of β-Arrestin 2 leads to increased mitogenic activity, decreased p21, and re-location of EGFR to the nucleus. This study reveals important tumor suppressor activity of β-Arrestin 2 in the context of EGFR signaling.

Unexpected Negative Role of the AT1R Adaptor Protein βarrestin1 in the ERK1/2 Activation of SHR‐Derived Primary Astrocytes

ObjectiveTo investigate the role of βarrestin1 in angiotensin II (AngII) type 1 receptor (AT1R)‐mediated extracellular signal‐regulated kinase (ERK1/2) activation in spontaneously hypertensive rat (SHR)‐derived primary astrocytes.BackgroundThe βarrestins, consisting of two isoforms, βarrestin1 and ‐2, originally discovered as terminators of G protein signaling by G protein‐coupled receptors (GPCRs), are now known to also mediate their own signaling to ERK1/2 activation independently of G proteins. The AT1R is a GPCR that can also signal to ERK1/2 via βarrestins in various cell types. However, whether it can do so in the brain has not been investigated yet. Thus herein, we sought to examine the involvement of βarrestins in the AT1R‐mediated ERK1/2 activation in SHR‐derived primary astrocytes.MethodsPrimary cultures of rat astrocytes from brainstem were isolated from the brains of 2–3 days old pups obtained from pregnant SHR. 100 nM AngII and 10 μM SII (an AngII peptide analog which is βarrestin‐biased agonist) were used to study the AT1R mediated ERK1/2 activation. The results were compared to normotensive Wistar rats. Protein analysis was done using Western blotting and mRNA measurement was done using real time PCR.ResultsOur results indicate that βarrestin1 is the major arrestin protein in astrocytes from both SHR and normotensive rats. At the mRNA level however, both βarrestin1 and βarrestin2 display comparable expression. AngII activates ERK1/2 in SHR astrocytes much more robustly than in normotensive rat ones. On the other hand, SII strongly activates ERK1/2 in normotensive rat astrocytes and only minimally in SHR ones. Importantly, AngII also upregulates βarrestin1 protein in normotensive rat astrocytes, whereas in SHR astrocytes it has no significant effect. Finally, AngII downregulates both βarrestins at the mRNA level in both SHR and normotensive rat astrocytes.ConclusionThe AngII‐AT1R‐induced ERK1/2 activation of SHR astrocytes, known to induce hypertension, appears to be almost exclusively G protein‐dependent, contrary to normotensive rat astrocytes. Since βarrestin1 protein is also upregulated by AngII in normotensive rat astrocytes and appears downregulated in SHR astrocytes, it is quite plausible that βarrestin1 may actually inhibit ERK1/2 activation in astrocytes. This might be a beneficial effect of βarrestin1 in preventing development of hypertension in the SHR condition.Support or Funding InformationFunding for this research was provided through a Nova Southeastern University Health Professions Division Grant

Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination

GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated.

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.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

(β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor-β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.

Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling.