GPCR Desensitization Research Articles

G proteins: introduction of its history, structure, function, and drug development

G protein-coupled receptor can be written in GPCR, it has a big great family and this species include 800 human genes, it become the important part of human body. Although each species has their own unique skills, it can make different kind of medicine that can save humans life. And then there's the G-protein-coupled receptor mechanism. The GPCR desensitization regulator - arrestin was further analyzed, and researchers discovered that GPCRs could be activated not only through the G-protein-dependent pathway but also through the non-G-protein-dependent pathway, known as the -inhibitor pathway, to control the ingestion and desensitization throughout vivo and even start a new wave of signal transduction. The development of G-protein-coupled receptor drugs followed. GPCR is strongly associated to pathological conditions and has an essential function in cell signal transmission. More than 40% of the medicines on the market today target GPCR, which is the reached a high point family of pharmacological targets. The intracellular effector proteins (G proteins, etc.) that are activated by the GPCR play an important role in the regulation of its physiological function.

Polycystin 1 Ciliary Localization Is Controlled by Chemo- and Mechanosensitive Signaling Processes That Involve the Machinery of GPCR Desensitization

Polycystin 1 Ciliary Localization Is Controlled by Chemo- and Mechanosensitive Signaling Processes That Involve the Machinery of GPCR Desensitization

The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs.

To support studies of neuropeptide neuromodulation, I have studied beta-arrestin binding sites (BBS's) by evaluating the incidence of BBS sequences among the C terminal tails (CTs) of each of the 49 Drosophila melanogaster neuropeptide GPCRs. BBS were identified by matches with a prediction derived from structural analysis of rhodopsin:arrestin and vasopressin receptor: arrestin complexes [1]. To increase the rigor of the identification, I determined the conservation of BBS sequences between two long-diverged species D. melanogaster and D. virilis. There is great diversity in the profile of BBS's in this group of GPCRs. I present evidence for conserved BBS's in a majority of the Drosophila neuropeptide GPCRs; notably some have no conserved BBS sequences. In addition, certain GPCRs display numerous conserved compound BBS's, and many GPCRs display BBS-like sequences in their intracellular loop (ICL) domains as well. Finally, 20 of the neuropeptide GPCRs are expressed as protein isoforms that vary in their CT domains. BBS profiles are typically different across related isoforms suggesting a need to diversify and regulate the extent and nature of GPCR:arrestin interactions. This work provides the initial basis to initiate future in vivo, genetic analyses in Drosophila to evaluate the roles of arrestins in neuropeptide GPCR desensitization, trafficking and signaling.

Deciphering the signaling mechanisms of β-arrestin1 and β-arrestin2 in regulation of cancer cell cycle and metastasis.

β-Arrestins are ubiquitously expressed intracellular proteins with many functions which interact directly and indirectly with a wide number of cellular partners and mediate downstream signaling. Originally, β-arrestins were identified for their contribution to GPCR desensitization to agonist-mediated activation, followed by receptor endocytosis and ubiquitylation. However, current investigations have now recognized that in addition to GPCR arresting (hence the name arrestin). β-Arrestins are adaptor proteins that control the recruitment, activation, and scaffolding of numerous cytoplasmic signaling complexes and assist in G-protein receptor signaling, thus bringing them into close proximity. They have participated in various cellular processes such as cell proliferation, migration, apoptosis, and transcription via canonical and noncanonical pathways. Despite their significant recognition in several physiological processes, these activities are also involved in the onset and progression of various cancers. This review delivers a concise overview of the role of β-arrestins with a primary emphasis on the signaling processes which underlie the mechanism of β-arrestins in the onset of cancer. Understanding these processes has important implications for understanding the therapeutic intervention and treatment of cancer in the future.

Abstract 166: Identification of Grk2 as novel oncology target and development of potent Grk2 inhibitors

Abstract G Protein-Coupled Receptor Kinase 2 (Grk2) is a serine-threonine kinase widely expressed in the human body. The GRK family of kinases is known for its role in GPCR phosphorylation and desensitization upon GPCR activation by agonists. Besides its role in GPCR phosphorylation, Grk2 has been shown to phosphorylate non-GPCR receptors and cytoplasmic substrates and has also been demonstrated to play phosphorylation-independent roles in cells. Using pooled CRISPR screening we identified that Grk2 knockout inhibits pancreatic and bladder cancer cell line proliferation in vitro and tumor growth in xenografts in vivo, which has not been demonstrated before. Rescue experiments re-expressing wildtype or kinase inactive Grk2 showed that this effect is dependent on catalytically active Grk2. We developed Grk2 inhibitors with exquisite enzymatic potency and anti-proliferative activity in pancreatic cancer cell lines. On a cellular level Grk2 knockout and Grk2 inhibitors slow down G2/M cell cycle progression in pancreatic cancer cell lines, and Grk2 inhibitor CYG-N-2278 induced a 71% tumor growth inhibition in PAXF1657 pancreatic PDX-derived xenografts. Besides its direct role in cancer cell growth Grk2 also plays a role in immune cell function. Grk2 is highly expressed in immune cells, and we have demonstrated that Grk2 inhibitors upregulate proinflammatory cytokine expression in myeloid cells. In the MC38 colorectal syngeneic tumor model implanted into immunocompetent C57BL6 mice CYG-N-2278 Grk2 inhibitor resulted in a 39% tumor growth inhibition, whereas no effect was observed on MC38 tumor growth in immunodeficient NSG mice, suggesting an immune cell mediated tumor growth inhibition. Analysis of The Cancer Genome Atlas data has showed that Grk2 is amplified across multiple tumor types, including bladder, uterine and cholangiocarcinoma cancer, and that Grk2 amplification is prognostic for tumor outcome, confirming the human relevance and suggesting a translatable patient selection strategy. In conclusion, we identified Grk2 as novel target for oncology, demonstrating a direct effect of Grk2 inhibition on cancer cells through inhibiting G2/M cell cycle progression and an indirect effect on tumor growth through immune cell activation. Cygnal’s Grk2 inhibitors possess superior potency over previous Grk2 inhibitors and have shown promising preclinical anti-tumor efficacy. Citation Format: Alexandra Lantermann, Eugene Chekler, Bruce Lefker, Garmen Yuen, Vanessa Lam, Matthew Strickland, Justyne Pennacchio, Aaron Fulgham, Dara Bree, Thomas Perekslis, Grazia Piizzi, Hongyue Dai, Tim Zheng, John Wagner, Pearl Huang. Identification of Grk2 as novel oncology target and development of potent Grk2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 166.

Role of arrestin1 in regulation of GPCR signaling in platelet activation

The arrestins are a class of soluble proteins that function in concert with the GPCR kinases to stop or arrest GPCR‐mediated intracellular signaling. Although β‐arrestins function in the desensitization of most GPCRs, the functional differences of β‐arrestin1 (arrestin2, BARR1) versus β‐arrestin2 (arrestin1, BARR2) in the regulation of GPCR signaling in platelets and the mechanisms of desensitization of platelets to GPCR agonists by β‐arrestins have not been understood. In this study, we identify the role of BARR2 in the regulation of platelet function and GPCR signaling in platelets. We used mice lacking BARR1 and BARR2 to evaluate the functional role of BARR2 in platelet activation. Platelet aggregation, dense‐granule secretion, and fibrinogen receptor activation induced by 2‐MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in BARR2 −/− platelets compared to WT platelets. In contrast to BARR2 −/− platelets, AYPGKF‐, thrombin‐, and U46619‐induced platelet aggregation and secretion were significantly inhibited in BARR1 −/− platelets compared to WT platelets, while 2‐MeSADP‐induced platelet aggregation and secretion were minimally affected in BARR1 −/−platelets. However, CRP‐induced platelet aggregation and secretion were not affected in both BARR1 −/− and BARR2 −/− platelets. These results suggest that BARR2 functions as the predominant regulator in the termination of GPCR signaling in platelets. In addition, platelet aggregation was restored in response to second challenge of 2‐MeSADP and AYPGKF in BARR2 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT and b‐arrestin1 −/− platelets, indicating BARR2 contributes to P2Y1, P2Y12, and PAR receptor desensitization. Furthermore, 2MeSADP‐ and AYPGKF‐induced Akt and ERK phosphorylations were significantly potentiated in BARR2 −/− platelets. Finally, BARR2 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating BARR2 is critical for thrombus formation in vivo. We conclude that BARR2 plays an important role in regulation of platelet function through selective GPCR desensitization.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).

Role of G protein‐coupled receptor kinase 6 in Regulation of Platelet Activation through Selective GPCR Desensitization

Many platelet agonists mediate their cellular effects through GPCRs to induce platelet activation. Considering the crucial roles of GPCR kinases (GRKs) in GPCR functions, little is known regarding the regulation of GPCR signaling by GRKs in platelets. In this study, we identify the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used mice lacking GRK6 to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense‐ and α‐granule secretion, and fibrinogen receptor activation induced by ADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6 −/− platelets compared to WT platelets. However, CRP‐induced platelet aggregation and secretion were not affected in GRK6 −/− platelets. Interestingly, platelet aggregation induced by co‐stimulation of serotonin and epinephrine which activate Gq‐coupled 5HT2A and Gz‐coupled α2A adrenergic receptor, respectively, was not affected in GRK6 −/− platelets, suggesting GRK6 is involved in specific GPCR regulation. In addition, platelet aggregation was restored in response to second challenge of ADP, U46619, thrombin, and AYPGKF in GRK6 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT platelets, indicating GRK6 contributes to P2Y1, P2Y12, thromboxane A2, and PAR receptor desensitization. The surface expression level of PAR4 upon AYPGKF stimulation in GRK6 −/− platelets was higher than WT platelets, indicating a crucial role of GRK6 in internalization of activated PAR4 receptor. Furthermore, AYPGKF‐induced Akt, ERK, and PKCδ phosphorylations were significantly potentiated and sustained in GRK6 −/− platelets. Finally, GRK6 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating GRK6 is critical for thrombus formation in vivo. We conclude that GRK6 plays an important role in regulating platelet functional responses through selective GPCR desensitization.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methods to Investigate β-Arrestin-Mediated Regulation of GPCR Function in Human Airway Smooth Muscle.

Arrestin proteins were originally characterized as regulators of GPCR desensitization, and that function alone was sufficient to promote extreme interest in their study. It is now appreciated that arrestins also function as mediators of GPCR trafficking and G protein-independent signaling. This latter function places them as prominent players in the emerging field of qualitative signaling, which promises to launch a new area of pharmacology that defines ligands with selectivity/bias toward either G protein-dependent or -independent signaling. To meet the demands of research into arrestin function, methodology has evolved accordingly over the last three decades since the discovery of the arrestin family. Herein we describe state-of-the-art approaches for studying the role of arrestins (β-arrestin1 aka arrestin 2, β-arrestin2 aka arrestin 3) in GPCR function in a primary cell type, cultured airway smooth muscle cells.

Species Differences in Visual Arrestin Multimerization Revealed by Analytical Ultracentrifugation

Arrestins play important roles in GPCR signaling and desensitization. The photoreceptor specific visual arrestin (Arr1) is a soluble protein with no known post-translational modifications, that is differently distributed throughout light and dark-adapted photoreceptor. Second only to its target GPCR, rhodopsin in concentration, mechanisms that underlie the translocation of such an abundant protein are not understood. Arr1 is predicted to self-associate up to tetramers across multiple mammalian species, varying mostly in the KD of the dimer and tetramer forms. One hypothesis on the localization of Arr1 in the cell body of dark-adapted photoreceptors is that the Arr1 tetramer is partitioned there due to differences in densities of subcellular structures in the two compartments and the impact of steric volume exclusion on the accessible volumes. To investigate whether this self-associative behavior applies across a broader range of species and other model organisms, we examined Xenopus laevis Arr1 (xArr1) which bears ∼68% sequence identity to mammalian Arr1s. Homology modeling based on the bovine crystal structure suggests that it is likely to have a very similar tertiary structure. Residues of mammalian Arr1 hypothesized to be at dimer and tetramer interfaces by their ability to affect the extent of oligomerization are mostly conserved in X. laevis as well, suggesting xArr1 would likely follow the same self-association pattern. We performed analytical ultracentrifugation experiments on xArr1 as well as bovine Arr1 (bArr1) for comparison. Our data for bArr1 agrees well with KDs from published studies, and the KD dimer for xArr1 fits well into the range published for human, mouse, and bovine Arr1. Surprisingly, at concentrations up to 210 micromolar, our results show xArr1 as monomer and dimer only, with no evidence of higher order oligomers. The mechanism underlying this difference is under investigation.

G‐protein Coupled Receptor Kinase‐2 Deficiency Protects Mice from Experimentally Induced Colitis

G‐protein coupled receptor kinase 2 (GRK2), a member of the serine/threonine family of GPCR kinases are known for their role in GPCR phosphorylation and desensitization. In addition to this canonical role, GRK2 also modulates inflammation via non‐canonical pathways and thus has been shown to be involved in some inflammatory diseases. However, role of GRK2 in inflammatory bowel disease is not known. We hypothesized that the ablation of GRK2 would significantly modulate the pathogenesis of dextran sodium sulfate (DSS)‐induced colitis in mice. To test this, we administered DSS (in drinking water) to GRK2 heterozygous (HET) and wild type (WT) mice for 6 days followed by drinking water for 1 day. We found that GRK2 HET mice were significantly protected from DSS‐induced colitis compared to WT mice. This was demonstrated by decreased weight loss (WT 20±2.8% vs HET 11±3.4% n=6–7), lower disease activity index scores (WT 9.1 vs HET 4.1; n=6–7), and increased colon lengths (WT 4.7 cm vs HET 5.3 cm; n=6–7) in the GRK2 HET compared to WT mice. Additionally, we observed significant decrease in inflammatory cytokines including IL‐17, GM‐CSF, as well as the IL‐6/IL‐10 ratio in the colon without significant differences in cellular infiltration at 7‐day post‐DSS treatment. To further identify the cellular mechanisms by which GRK2 modulates colitis, we investigated the role for GRK2 in a myeloid‐specific GRK2 knockout. Our results demonstrate that GRK2 deficiency in the myeloid cells is sufficient for the protection observed in the heterozygous mice. Together our results indicate that GRK2 is an important mediator of colitis pathogenesis and this is conferred by the myeloid‐specific GRK2. Our results further suggest that GRK2 could be an important therapeutic target in colitis.Support or Funding InformationFunding provided through NIH Grants 4R01AT007695‐04, 5R01DK101050‐03, 5R21AI099404‐02

CAMP assays in GPCR drug discovery.

GPCRs are a major family of cell surface receptors and the most druggable protein targets in modern medicine. Recent elucidation of crystal structures of a vast number of GPCRs eludes to the finer details of biased agonism or functional selectivity of many of these receptors and warrants a better understanding of their biological effects as well as therapeutic potential. Receptor function is measured in terms desensitization/resensitization, which provides insights on receptor activation and differential coupling to various G proteins. This review article presents thoughts on the potential of GPCR desensitization assay/cAMP assay, a highly sensitive and versatile platform for high-throughput assays for elucidating novel functions of many orphan GPCRs. Besides, these assays also are very sensitive to screen for agonists, partial agonists, or antagonists and provide a simplified system for novel drug discovery for Gs-coupled GPCRs and understanding their physiological implications.

Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2’s kinase activity

BackgroundAmphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methodsβ2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization. ResultsMembrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization. ConclusionAmphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General SignificanceCommonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2’s kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

Dopamine receptors - IUPHAR Review 13.

The variety of physiological functions controlled by dopamine in the brain and periphery is mediated by the D1, D2, D3, D4 and D5 dopamine GPCRs. Drugs acting on dopamine receptors are significant tools for the management of several neuropsychiatric disorders including schizophrenia, bipolar disorder, depression and Parkinson's disease. Recent investigations of dopamine receptor signalling have shown that dopamine receptors, apart from their canonical action on cAMP-mediated signalling, can regulate a myriad of cellular responses to fine-tune the expression of dopamine-associated behaviours and functions. Such signalling mechanisms may involve alternate G protein coupling or non-G protein mechanisms involving ion channels, receptor tyrosine kinases or proteins such as β-arrestins that are classically involved in GPCR desensitization. Another level of complexity is the growing appreciation of the physiological roles played by dopamine receptor heteromers. Applications of new in vivo techniques have significantly furthered the understanding of the physiological functions played by dopamine receptors. Here we provide an update of the current knowledge regarding the complex biology, signalling, physiology and pharmacology of dopamine receptors.

Novel roles for β‐arrestins in the regulation of pharmacological sequestration to predict agonist‐induced desensitization of dopamine D3 receptors

In addition to typical GPCR kinase (GRK)-/β-arrestin-dependent internalization, dopamine D3 receptor employed an additional GRK-independent sequestration pathway. In this study, we investigated the molecular mechanism of this novel sequestration pathway. Radioligand binding, flow cytometry and cell surface biotinylation assay were used to characterize trafficking properties of D2 and D3 receptors. Serine/threonine and N-linked glycosylation mutants of the D3 receptor were utilized to locate receptor regions involved in pharmacological sequestration and desensitization. Various point mutants of the D2 and D3 receptors, whose sequestration and desensitization properties were altered, were combined with knockdown cells of GRKs or β-arrestins to functionally correlate pharmacological sequestration and desensitization. The D3 receptor, but not the D2 receptor, showed characteristic trafficking behaviour in which receptors were shifted towards the more hydrophobic domains within the plasma membrane without translocation into other intracellular compartments. Among various amino acid residues tested, S145/S146, C147 and N12/19 were involved in pharmacological sequestration and receptor desensitization. Both pharmacological sequestration and desensitization of D3 receptor required β-arrestins, and functional relationship was observed between two processes when it was tested for D3 receptor variants and agonists. Pharmacological sequestration of D3 receptor accompanies movement of cell surface receptors into a more hydrophobic fraction within the plasma membrane and renders D3 receptor inaccessible to hydrophilic ligands. Pharmacological sequestration is correlated with desensitization of the D3 receptor in a Gβγ- and β-arrestin-dependent manner. This study provides new insights into molecular mechanism governing GPCR trafficking and desensitization.

Identification of a Novel Protein-Protein Interaction Motif Mediating Interaction of GPCR-Associated Sorting Proteins with G Protein-Coupled Receptors

GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.

Roles of GRK2 in Cell Signaling Beyond GPCR Desensitization: GRK2-HDAC6 Interaction Modulates Cell Spreading and MotilityA Presentation from the Cell Signaling Networks Conference and 13th IUBMB Conference, Mérida, Yucatán, México, 22 to 27 October 2011.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous, essential protein kinase that is emerging as an integrative node in many signaling networks. Moreover, changes in GRK2 abundance and activity have been identified in several inflammatory, cardiovascular disease, and tumor contexts, suggesting that those alterations may contribute to the initiation or development of pathologies. GRKs were initially identified as key players in the desensitization and internalization of multiple G protein-coupled receptors (GPCRs), but GRK2 also phosphorylates several non-GPCR substrates and dynamically associates with a variety of proteins related to signal transduction. Ongoing research in our laboratory is aimed at understanding how specific GRK2 interactomes are orchestrated in a stimulus-, context-, or cell type-specific manner. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6) that modulates cell spreading and motility. HDAC6 is a major cytoplasmic a-tubulin deacetylase that is involved in cell motility and adhesion. GRK2 dynamically and directly associates with and phosphorylates HDAC6 to stimulate its a-tubulin deacetylase activity at specific cellular localizations, such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. GRK2-HDAC6-mediated regulation of tubulin acetylation also modulates cellular spreading. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to epithelial cell migration.

Novel Roles for the E3 Ubiquitin Ligase Atrophin-interacting Protein 4 and Signal Transduction Adaptor Molecule 1 in G Protein-coupled Receptor Signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

Insulin Action under Arrestin

Insulin signaling is key to the etiology of metabolic syndrome. Recent work (Luan et al., 2009) uncovers a role for beta-arrestin, previously known to control GPCR desensitization, in insulin signaling. In mouse models, beta-arrestin-2 controls whole-body insulin action by regulating assembly of a complex containing insulin receptor, c-Src, and Akt.

Functional characterization of kurtz, a Drosophila non-visual arrestin, reveals conservation of GPCR desensitization mechanisms

The arrestins are a family of molecules that terminate signaling from many different G protein-coupled receptors, by inhibiting the association between receptor and downstream effectors. We recently employed a human βarrestin2-GFP fusion protein to explore the dynamics of different neuropeptide receptors in Drosophila and have previously used a βarrestin translocation assay to identify ligands at orphan receptors. Here, we report that the Drosophila arrestin encoded by kurtz functions in a similar fashion and can be employed to investigate GPCR–arrestin associations. Specifically, a GFP– krz fusion protein, upon co-expression with various Drosophila peptide receptors, an amine receptor, and a mammalian peptide receptor translocates to the plasma membrane in specific response to ligand application. This molecular phenotype is exhibited in a mammalian cell line as well as in a Drosophila cell line. Notably, the details of receptor–arrestin associations in terms of endocytotic patterns are functionally conserved between the mammalian arrestins and kurtz. Furthermore, we report that kurtz mutants exhibit hypersensitivity to osmotic stress, implicating GPCR desensitization as an important feature of the endocrine events that shape this stress response.

Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (-/-) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3-/- mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3-/- neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR-/-, or GRK3-/- mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.