Beta-Arrestins

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HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Novel findings reveal important functional roles for β-arrestin 1 and β-arrestin 2 in the regulation of insulin secretion, β-cell survival, and -cell mass plasticity not only by glucose, but also by G-protein coupled receptors, such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors or GPR40, or tyrosine kinase receptors, such as the insulin receptor. Here,we describe experimental protocols to knockdown -arrestins by small interference RNA, to follow subcellular localization of arrestins in the cytosol and nucleus of the insulinoma INS-1E rat pancreatic -cell line, to analyse -arrestin protein expression by western blot using INS-1E cells and isolated mouse or human pancreatic islets. We also provide details on how to genotype -arrestin 2 knockout (Arrb2 -/-) mice, and to evaluate β-arrestin-mediated roles in β-cell mass plasticity and β-cell signalling using immunocytochemistry on pancreatic sections or on primary dispersed β-cells from wild type mice and Arrb2 -/-mice.

4-cresol (4-methylphenol, p-cresol) is a xenobiotic substance negatively correlated with type 2 diabetes and associated with health improvement in preclinical models of diabetes. We aimed at refining our understanding of the physiological role of this metabolite and identifying potential signalling mechanisms. Functional studies revealed that 4-cresol does not deteriorate insulin sensitivity in human primary adipocytes and exhibits an additive effect to that of insulin on insulin sensitivity in mouse C2C12 myoblasts. Experiments in mouse isolated islets showed that 4-cresol potentiates glucose induced insulin secretion. We demonstrated the absence of off target effects of 4-cresol on a panel of 44 pharmacological compounds. Screening large panels of 241 G protein-coupled receptors (GPCRs) and 468 kinases identified binding of 4-cresol only to TNK1, EIF2AK4 (GCN2) and RPS6KA3 (RSK2), a kinase strongly expressed in human and rat pancreatic islets. Islet expression of RPS6KA3 is reduced in spontaneously diabetic rats chronically treated with 4-cresol and Rps6ka3 deficient mice exhibit reduction in both body weight and fasting glycemia, modest improvement in glycemic control and enhanced insulin release in vivo. Similar to low doses of 4-cresol, incubation of isolated rat islets with low concentrations of the RPS6KA3 inhibitor BIX 02565 stimulates both glucose induced insulin secretion and β-cell proliferation. These results provide further information on the role of low 4-cresol doses in the regulation of insulin secretion.

Background
Neuroinflammation is one of the features observed in neurodegenerative diseases, and inflammatory cytokines and chemokines are two of the important players in the neuroinflammatory responses. β-arrestin 2 has also been reported to play a crucial role in regulating cytokines and chemokines release as the loss of β-arrestin 2 leads to an increase in the sepsis-induced inflammation. However, the role of β-arrestin 2 in regulating the TNF mediated cytokines and chemokines production is poorly understood.

Method
Stable β-arrestin 2 knockdown (KD) and negative control (NC) SH-SY5Y clone cells were used for this study. Subcellular fractionation was used to investigate the cell surface TNF-R1, and co-immunoprecipitation (co-IP) was performed to determine for the possible interaction between TNF-R1 and β-arrestin 2. Immunoblotting was used to investigate P65 phosphorylation status. Lastly, cytokines and chemokines mRNA expressions were quantified using Real Time-PCR after TNF treatment.

Result
Subcellular fractionation showed significantly more TNF-R1 remained on the cell membrane fraction of KD compared to NC. Using NC cells, co-IP showed the possible interaction between TNF-R1 and β-arrestin 2. KD also demonstrated significant increase in P65 phosphorylation compared to NC, along with significantly higher mRNA expressions of cytokines and chemokines.

Conclusion
These results suggest that prolong TNF-R1 on the membrane, possibly due to the lack of desensitization by β-arrestin 2, may have led to an increase in the P65 phosphorylation, and resulting in a significant increase in the cytokines and chemokines mRNA expressions. Hence, our findings established the important role of β-arrestin 2 in the TNF mediated NFκB pathway, and cytokines and chemokines production.

β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of G s -dependent β 1 AR and G i -dependent β 2 AR pathways leads to enhanced cardiomyocyte death, reduced β 1 AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β 1 AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, β-arrestin-dependent signaling promotes cardiomyocyte survival. Given that β 2 AR expression is unaltered in CHF, a β-arrestin-biased agonist that operates through the β 2 AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin-biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin-biased pepducin for the β 2 AR, intracellular loop (ICL)1-9, was used to decouple β-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote β 2 AR phosphorylation, β-arrestin recruitment, β 2 AR internalization, and β-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce β 2 AR-and β-arrestin-dependent and Ca 2+ -independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β 2 AR and serves as a model for the next generation of cardiovascular drug development.

β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent β1AR and Gi-dependent β2AR pathways leads to enhanced cardiomyocyte death, reduced β1AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, β-arrestin-dependent signaling promotes cardiomyocyte survival. Given that β2AR expression is unaltered in CHF, a β-arrestin-biased agonist that operates through the β2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin-biased agonist t...

Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cellbased functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.

II has many biological effects in renal physiology, particularly in Ca 2ϩ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine reninangiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo) plasmic reticulum Ca 2ϩ -ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrinindependent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ␤-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ␤-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKC␣ in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca 2ϩ responses. ANG II-AT1R/AT2R complex; LLC-PK1 luminal membranes; ANG II-AT1R/AT2R internalization; endoplasmic reticulum; microtubuledependent clathrin-independent endocytosis THE RENIN-ANGIOTENSIN SYSTEM (RAS) is a key hormonal signaling system involved in the homeostasis of body salt and fluid, operating mainly at the kidney level, where the best-described active peptide, ANG II, is the main regulator of Na ϩ and water reabsorption, a function that depends on finely tuned Ca 2ϩ transport in the cell (6, 15).

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.

G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and important targets of drug action. Of the approximately 350 non-olfactory human GPCRs, more than 100 are still considered "orphans" as their endogenous ligand(s) remain unknown. Here, we describe a unique open-source resource that provides the capacity to interrogate the druggable human GPCRome via a G protein-independent β-arrestin recruitment assay. We validate this unique platform at more than 120 non-orphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs, and describe a method (PRESTO-TANGO; Parallel Receptorome Expression and Screening via Transcriptional Output-TANGO) for the simultaneous and parallel interrogation of the entire human GPCR-ome. G protein-coupled receptors (GPCRs) are proteins with seven transmembrane helices capable of transducing a wide variety of extracellular stimuli into intracellular signals, mediated by G proteins of four groups, G s , G i , G 12 or 13 or G q , as well as arrestins and other effectors 1. The human genome encodes more than 350 different non-olfactory GPCRs, as well as a similar number of olfactory GPCRs 2-4. In addition to their roles as signal transducers, GPCRs are the targets for more than one-third of currently prescribed medications 5, 6. Approximately one-third of the non-olfactory GPCRs in the human genome are "orphan" GPCRs, i.e., their endogenous or natural ligands are unknown 2-4 , while many more are inadequately interrogated with respect to their ligands. Thus, much of the druggable GPCR-ome-like other drug target families such as the kinome 7 , represents 'dark Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)–endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial–mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, an...

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor ...

Objective In chronic inflammation, prevention of cAMP degradation by phosphodiesterase-4 (PDE4) inhibition can be anti-inflammatory therapy. However, PDE4 inhibition was uneffective in rheumatoid arthritis (RA). Recent studies demonstrated that PDE4/β-arrestin interaction at β-adrenoceptors resulted in switching from Gαs to Gαi signaling and ERK1/2 activation. Such a switch in signaling might elicit proinflammatory effects. We aimed to investigate this possible Gαs to Gαi signaling switch in RA and osteoarthritis (OA) mixed synoviocytes. Methods Synoviocytes were treated alone or with combinations of adrenergic, dopaminergic, and adenosinergic drugs, rolipram (PDE4 inhibitor), inhibitors of Gαi signaling (pertussis toxin), and blockers of protein kinase A (PKA). Under normoxic or hypoxic conditions, proinflammatory TNF was the readout-parameter. We investigated co-expression and interaction of PDE4 and β-arrestin by imaging techniques. Expression of pERK1/2 was analyzed by western blotting. Results Mixed synoviocytes in RA and OA possessed all major Gαs-coupled neurotransmitter receptors. Under hypoxia, particularly in RA cells, Gαs-coupled receptor agonists unexpectedly increased TNF and respective antagonists decreased TNF. Under hypoxia, rolipram alone or rolipram plus Gαs agonists increased TNF, which was reversed by pertussis toxin or PKA inhibition. Colocalization and interaction of PDE4 and β-arrestin in synovial tissue and cells was demonstrated. Gαs agonists or rolipram plus Gαs agonists increased pERK1/2 expression. 3 Conclusions This study in human arthritic synovial tissue presents an unexpected proinflammatory switch from Gαs to Gαi signaling, which depends on PDE4/βarrestin interaction. This phenomenon is most probably responsible for reduced efficacy of PDE4 inhibitors and Gαs agonists in RA.

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and β-arrestin- (βArr) mediated signaling. To separate these signaling modalities, we have used βArr1 and βArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and βArr1-KO mice, without effects in βArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and βArr1-KO animals. By contrast, in βArr2-KO mice head twitch responses are low with LSD and this psychedelic is without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks the LSD e...

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)–endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial–mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, an...

Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold β-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We pr...

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5 + endosomes as serine-phosphoproteins bound to β-arrestin. This results from the ability of p66Shc to inhibit Ca 2+ and PP2B-dependent CXCR4/CCR7 dephosphorylation and β-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.

Many interaction partners of β-arrestins intervene in the control of mRNA translation. However, how β-arrestins regulate this cellular process has been poorly explored. In this study, we show that β-arrestins constitutively assemble a p70S6K/ribosomal protein S6 (rpS6) complex in HEK293 cells and in primary Sertoli cells of the testis. We demonstrate that this interaction is direct, and experimentally validate the interaction interface between β-arrestin 1 and p70S6K predicted by our docking algorithm. Like most GPCRs, the biological function of follicle-stimulating hormone receptor (FSHR) is transduced by G proteins and β-arrestins. Upon follicle-stimulating hormone (FSH) stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the β-arrestin/p70S6K/rpS6 preassembled complex, which is not recruited to the FSHR. In agreement, FSH-induced rpS6 phosphorylation within the β-arrestin scaffold was decreased in cells depleted of Gαs Integration of the coope...

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antago...