Β-arrestin Signaling Research Articles

A cross-platform approach identifies genetic regulators of human metabolism and health.

In cross-platform analyses of 174 metabolites, we identify 499 associations (P < 4.9 × 10-10) characterized by pleiotropy, allelic heterogeneity, large and nonlinear effects and enrichment for nonsynonymous variation. We identify a signal at GLP2R (p.Asp470Asn) shared among higher citrulline levels, body mass index, fasting glucose-dependent insulinotropic peptide and type 2 diabetes, with β-arrestin signaling as the underlying mechanism. Genetically higher serine levels are shown to reduce the likelihood (by 95%) and predict development of macular telangiectasia type 2, a rare degenerative retinal disease. Integration of genomic and small molecule data across platforms enables the discovery of regulators of human metabolism and translation into clinical insights.

Pharmacological Characterization of µ-Opioid Receptor Agonists with Biased G Protein or β-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation.

In recent years, G protein vs. β-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the β-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure β-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand–receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or β-arrestin.

GLP‐1 modulates insulin‐induced relaxation response through β‐arrestin2 regulation in diabetic mice aortas

Diabetes impairs insulin-induced endothelium-dependent relaxation by reducing nitric oxide (NO) production. GLP-1, an incretin hormone, has been shown to prevent the development of endothelial dysfunction. In this study, we hypothesized that GLP-1 would improve the impaired insulin-induced relaxation response in diabetic mice. We also examined the underlying mechanisms. Using aortic rings from ob/ob mice, an animal model of obesity and type 2 diabetes, and from lean mice, vascular relaxation responses and protein expressions were evaluated using insulin, GLP-1, and pathway-specific inhibitors to elucidate the mechanisms of response. In parallel experiments, β-arrestin2 siRNA-transfected aortas were treated with GLP-1 to evaluate its effects on aortic response pathways. When compared to that of untreated ob/ob aortas, GLP-1 increased insulin-induced vasorelaxation and NO production. AMPK inhibition did not alter this vasorelaxation in both GLP-1-treated lean and ob/ob aortas, while Akt inhibition reduced vasorelaxation in both groups, and co-treatment with GLP-1 and insulin caused Akt/eNOS activation. Additionally, GLP-1 decreased GRK2 activity and enhanced β-arrestin2 translocation from the cytosol to membrane in ob/ob aortas. β-Arrestin2 siRNA decreased insulin-induced relaxation both in lean aortas and GLP-1-treated ob/ob aortas. We demonstrated that insulin-induced relaxation is dependent on β-arrestin2 translocation and Akt activation via GLP-1-stimulated GRK2 inactivation in ob/ob aortas. We showed a novel cross-talk between GLP-1-responsive β-arrestin2 and insulin signalling in diabetic aortas.

Abstract 15386: Integrated Phosphoproteome and Transcriptome Analysis of Biased Agonists at CXCR3 Reveals Differential Cellular Outputs of Inflammation

Background and Objective: G-protein-coupled receptors (GPCRs) are the most common cell surface receptor class and are known to influence nearly every biological process. GPCRs signal through downstream effectors such as G proteins and β-arrestins. Certain ligands drive activation of G protein over β-arrestin signaling, or vice versa - a phenomenon known as biased agonism. To investigate the mechanisms and implications of biased agonism, we studied CXCR3, a chemokine receptor expressed on T cells, which binds three biased endogenous ligands, CXCL9, CXCL10, and CXCL11. Importantly, CXCR3 has been implicated in atherosclerosis and other inflammatory diseases. Methods: To obtain a broad view of biased signaling at CXCR3, we performed global phosphoproteomic and transcriptomic analyses of CXCR3-transfected HEK 293 cells and activated human CD8+ T-cells, respectively, treated with CXCL9, CXCL10, and CXCL11. To assess the phosphoproteome, we utilized an antibody-free mass spectrometry approach with high-pressure, high-resolution separation and intelligent selection. RNA-seq was performed to assess transcriptomic changes in activated CD8+ T-cells. Results: Our analyses revealed 1532 phosphosites and 887 transcripts differentially regulated between treatment groups. Gene Set Enrichment Analysis (GSEA) of CXCL11-treated cells revealed enrichment of pro-inflammatory targets including the Myc and TNF-α pathways via NFKB not observed in CXCL10- or CXCL9-treated cells. GSEA of CXCL10- and CXCL11-treated cells also revealed enrichment of the IL6-JAK-STAT3 pathway, linked to chronic inflammatory conditions. Kinase-Substrate Enrichment Analysis (KSEA) revealed differential phosphorylation activity associated with multiple kinases including AURKB (CXCL9), ROCK1 (CXCL9, 10, 11), CDK9 (CXCL10), and ATM (CXCL10, 11) - all known to regulate pro-inflammatory targets. Several phosphoproteins were transcription factors, including p53, known to mediate IL-6 production and atherosclerosis pathogenesis. Conclusion: Together, our integrated analysis of phosproteomic and transcriptomic data reveal the extent of differential cellular outputs produced by biased agonists at CXCR3 in modulating targets implicated in inflammatory diseases.

The antidepressant-like activity of chiral xanthone derivatives may be mediated by 5-HT1A receptor and β-arrestin signalling.

Our previous studies showed that xanthone derivatives with N-(2-methoxyphenyl)piperazine fragment have an affinity to the 5-HT1A receptor and show antidepressant-like properties in rodents. In this study, we tested three xanthone derivatives, HBK-1 (R, S) and its enantiomers, in which we increased the distance between the piperazine and xanthone fragments by using a hydroxypropoxy linker. We hypothesized that this would increase the binding to the 5-HT1A receptor and consequently, pharmacological activity. We aimed to assess the in vitro and in vivo pharmacological activity of the xanthone derivatives. We evaluated the in vitro affinity for serotonin 5-HT1A and 5-HT2A receptors and serotonin transporter. We also determined the intrinsic activity at the 5-HT1A receptor. We investigated the antidepressant-like properties and safety after acute administration (dose range: 1.25-20 mg/kg) using the forced swim, tail suspension, locomotor activity, rotarod and chimney tests in mice. We also evaluated the basic pharmacokinetic parameters. Our results indicated that the compounds showed a high affinity for the 5-HT1A receptor but very weak antagonistic properties in the Ca2+ mobilization assay; however, they showed significant agonistic properties in the β-arrestin recruitment assay. In both behavioural tests the studied xanthone derivatives showed antidepressant-like activity. Pre-treatment with p-chlorophenylalanine or WAY-100635 abolished their antidepressant-like activity. None of the compounds caused motor impairments at antidepressant-like doses. The racemate penetrated the blood-brain barrier and had a relatively high bioavailability after intraperitoneal administration. Xanthone derivatives with N-(2-methoxyphenyl)piperazine fragment and hydroxypropoxy linker show increased binding to the 5-HT1A receptor and may represent an attractive putative treatment candidate for depression.

β-Arrestin as a Therapeutic Target in Heart Failure.

Heart failure is a major source of morbidity and mortality, driven, in part, by maladaptive sympathetic hyperactivity in response to poor cardiac output. Current therapies target β-adrenergic and angiotensin II G protein-coupled receptors to reduce adverse cardiac remodeling and improve clinical outcomes; however, there is a pressing need for new therapeutic approaches to preserve cardiac function. β-arrestin is a multifunctional protein which has come under analysis in recent years as a key player in G protein-coupled receptor signal transduction and a potential therapeutic target in heart failure. β-arrestin attenuates β-adrenergic and angiotensin II receptor signaling to limit the deleterious response to excessive sympathetic stimulation while simultaneously transactivating cardioprotective signaling cascades that preserve cardiac structure and function in response to injury. β-arrestin signaling may provide unique advantages compared to classic heart failure treatment approaches, but a number of challenges currently limit clinical applications. In this review, we discuss the role and functions of β-arrestin and the current attempts to develop G protein-coupled receptor agonists biased towards β-arrestin activation. Furthermore, we examine the functional diversity of cardiac β-arrestin isotypes to explore key considerations in the promise of β-arrestin as a pharmacotherapeutic target in heart failure.

Mu Opioids Induce Biased Signaling at the Full-Length Seven Transmembrane C-Terminal Splice Variants of the mu Opioid Receptor Gene, Oprm1.

The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and β-arrestin2 signaling pathways. The concept that the G protein pathway is often linked to the therapeutic effect of the drug, while the β-arrestin pathway is associated to the side effects has been proposed to develop biased analgesic compounds with limited side-effects associated with traditional opiates. The mu opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms that are conserved from rodent to human. One type of the Oprm1 splice variants are the full-length 7 transmembrane (7TM) C-terminal splice variants, which have identical receptor structures including entire binding pocket, but contain a different intracellular C-terminal tail resulted from 3' alternative splicing. Increasing evidence suggest that these full-length 7TM C-terminal variants play important roles in mu opioid pharmacology, raising questions regarding biased signaling at these multiple C-terminal variants. In the present study, we investigated the effect of different C-terminal variants on mu agonist-induced G protein coupling, β-arrestin2 recruitment, and ultimately, signaling bias. We found that mu agonists produced marked differences in G protein activation and β-arrestin2 recruitment among various C-terminal variants, leading to biased signaling at various level. Particularly, MOR-1O, an exon 7-associated variant, showed greater β-arrestin2 bias for most mu agonists than MOR-1, an exon 4-associated variant. Biased signaling of G protein-coupled receptors has been defined by evidences that different agonists can produce divergent signaling transduction pathways through a single receptor. Our findings that a single mu agonist can induce differential signaling through multiple 7TM splice variants provide a new perspective on biased signaling at least for Oprm1, which perhaps is important for our understanding of the complex mu opioid actions in vivo where all the 7TM splice variants co-exist.

A non–GPCR-binding partner interacts with a novel surface on β-arrestin1 to mediate GPCR signaling

The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin-dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling.

Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

Loss of APJ mediated β-arrestin signalling improves high-fat diet induced metabolic dysfunction but does not alter cardiac function in mice.

Apelin receptor (APJ) is a G protein-coupled receptor that contributes to many physiological processes and is emerging as a therapeutic target to treat a variety of diseases. For most disease indications the role of G protein vs β-arrestin signalling in mitigating disease pathophysiology remains poorly understood. This hinders the development of G protein biased APJ agonists, which have been proposed to have several advantages over balanced APJ signalling agonists. To elucidate the contribution of APJ β-arrestin signalling, we generated a transgenic mouse harbouring a point mutation (APJ I107A) that maintains full G protein activity but fails to recruit β-arrestin following receptor activation. APJ I107A mutant mice did not alter cardiac function at rest, following exercise challenge or in response to pressure overload induced cardiac hypertrophy. Additionally, APJ I107A mice have comparable body weights, plasma glucose and lipid levels relative to WT mice when fed a chow diet. However, APJ I107A mice showed significantly lower body weight, blood insulin levels, improved glucose tolerance and greater insulin sensitivity when fed a high-fat diet. Furthermore, loss of APJ β-arrestin signalling also affected fat composition and the expression of lipid metabolism related genes in adipose tissue from high-fat fed mice. Taken together, our results suggest that G protein biased APJ activation may be more effective for certain disease indications given that loss of APJ mediated β-arrestin signalling appears to mitigate several aspects of diet induced metabolic dysfunction.

Drd2 biased agonist prevents neurodegeneration against NLRP3 inflammasome in Parkinson’s disease model via a β-arrestin2-biased mechanism

Activated astrocytes secrete inflammatory cytokines such as interleukin-1β (IL-1β) into the extracellular milieu, damaging surrounding neurons and involving in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). Dopamine receptor D2 (Drd2) expresses both in neurons and astrocytes, and neuronal Drd2 is a significant target in therapy of PD. Our previous study reveals that astrocytic Drd2 exerts anti-inflammatory effect via non-classical β-arrestin2 signaling in PD model. Therefore, seeking new biased ligands of Drd2 with better efficacy and fewer side effects to treat PD is desirable and meaningful. In the present study, we evaluated the effects of UNC9995, a novel biased Drd2 agonist on astrocyte-derived neuroinflammation and dopaminergic (DA) neuron degenerationin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We showed that UNC9995 rescued the TH+ neurons loss and inhibited glial cells activation in mouse substantia nigra in a Drd2 dependent manner. Focusing on astrocytes, we found UNC9995 shows a relatively safe concentration range and significantly suppresses astrocytic NLRP3 inflammasome activation induced by lipopolysaccharide plus ATP. Further study revealed that the anti-inflammatory effect of UNC9995 is independent of Drd2 / Gαi protein pathway. It activates β-arrestin2 by recruiting it to cell membrane. Critically, UNC9995 enhances β-arrestin2 interacting with NLRP3 to interfere inflammasome assembly, which consequently reduces IL-1β production. On the other hand, UNC9995 inhibits IL-1β-induced inflammatory pathway activation in DA neurons and rescues subsequent apoptosis via β-arrestin2 interacting with protein kinases, such as JNK and suppressing their phosphorylation. Furthermore, β-arrestin2 knockout abolishes the anti-inflammatory and neuroprotective effects of UNC9995 in PD mouse model, supporting that UNC9995 is a β-arrestin2-biased Drd2 agonist and revealing its novel function in PD treatment. Collectively, this work illustrates that Drd2 agonist UNC9995 prevents DA neuron degeneration in PD and provides a new strategy for developing the β-arrestin2-biased ligands in the therapy of NDDs.

Abstract 417: Cardiac Akap12 Signalosome Overexpression Exacerbation of Heart Failure and β-arrestin Signaling Pathway

Background: Heart failure (HF) is responsible for 1 out of 8 deaths per year in the U.S.A. andis the major cause of death globally. In HF, chronic β-adrenergic receptor (β-AR)stimulation leads to reduced cardiac function due in part to β-AR desensitization.β-arrestins are proteins that play a major role in desensitizing G protein-coupledreceptors (GPCRs) such as β-AR. Previously we reported enhanced cardiacfunction in mice lacking functional A Kinase Anchoring Protein 12 (AKAP12). Inthis study, we aim to investigate the impact of AKAP12 overexpression(oxAKAP12) on HF progression through assessing β-arrestins. Our central hypothesis is that cardiac AKAP12 overexpression potentiates HF developmentby influencing β-arrestin signaling downstream of the β-AR. Methods: HF was developed in WT and oxAKAP12-Tg mice (8-10) weeks old males andfemales, through chronic Isoproterenol (ISO) administration (60mg/kg/day for 14days). Left ventricular homogenates were used for gene expression analysis.Furthermore, AKAP12 was transiently overexpressed in AC16 cells (humancardiomyocytes cell line), to asses protein expression levels and Gαs pathwayactivity, upon treatment with 100 nM of ISO. Results: Cardiac oxAKAP12 in both males and females reduced left ventricular ejectionfraction (EF) by 14.5±2.5% and fractional shortening (FS) by 22.7±2% after 14-days of chronic ISO treatment when compared to control groups. β-arrestin-1gene expression levels were significantly lower (p=0.022) in oxAKAP12 malehomogenates treated with ISO (14 days) compared to control groups.Interestingly, female homogenates overexpressing AKAP12 showed significantlyhigher β-arrestin-1 gene expression levels (p&lt;0.0001) with ISO treatment,compared to control groups. In AC16 cells overexpressing AKAP12 and treatedwith ISO, Gαs activity and β-arrestin-1 protein expression levels were bothreduced by 50% compared to AC16 ISO treated groups. Conclusion: Cardiac oxAKAP12 negatively influences systolic function in both male andfemale mice, potentially through affecting β-arrestin signaling pathway. Thus,designing novel drugs to inhibit AKAP12 is promising to ameliorate HF.

Individual phosphorylation sites at the C-terminus of the apelin receptor play different roles in signal transduction

The apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls apelin receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of β-arrestins. We sought to determine the precise mechanisms by which apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of β-arrestin phosphorylation on G-protein-coupled receptor (GPCR)/β-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJ-mediated G-protein-dependent and β-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in β-arrestin1 and three in β-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in β-arrestin1 and Tyr48 in β-arrestin2. APJ mutations did not affect the phosphorylation of β-arrestins, but it affects the β-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the receptor to interact with β-arrestin1/2 and AP2, indicating that APJ affects the β-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the receptor to interact with GRK2 and β-arrestin1/2 upon stimulation with apelin-36, and disrupted receptor internalization and β-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases.

Therapeutic effect of omega-3 fatty acids on T cell-mediated autoimmune diseases.

The present study was to demonstrate that the G protein coupled receptors serve as targets for the treatment of autoimmune disease such as rheumatoid arthritis and multiple sclerosis. Rats received pristane at the base of the tail. Affected joints were counted daily. The T cell mediated autoimmune diseases such as pristine-induced arthritis (PIA) and autoimmune encephalomyelitis (EAE) in a rat model were profoundly ameliorated by treatment with the specific G protein couple receptors 120 (GPR120) stimuli omega-3 fatty acids (ω-3 FAs). Our study further revealed that the activation of GPR120 by ω-3 FAs can result in a decrease of phosphorylated transforming growth factor-β activated kinase 1 (TAK1), and further inhibit the downstream IKKβ/I-κB pathway and the terminal NF-κB activation which serves as a mediator of T cell activation. ω-3 Fatty acids exhibited an inhibitory effect on TAK1 by enhancing the association of β-arrestin2 and TAK1 binding protein 1 (TAB1), thus the disassociation of TAB1 from the TAB1/TAK1 complex renders a limited effect on β-arrestin2 signaling as an innate immunity regulation. GPR120 is a functional receptor of ω-3 fatty acids in T cell-mediated autoimmune disease compared with its effect on innate immunity.

A G protein-biased S1P1 agonist, SAR247799, protects endothelial cells without affecting lymphocyte numbers.

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P1) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P1, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P1-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P1 These findings demonstrate that sustained S1P1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.

Molecular mechanisms of fentanyl mediated β-arrestin biased signaling

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or β-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent β-arrestin biased agonist, binds the μ-opioid receptor (μOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. Following an in-silico derived hypothesis we found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce β-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate an activation mechanism where the n-aniline ring of fentanyl mediates μOR β-arrestin through a novel M153 "microswitch" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated β-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.

Etrasimod, a Selective S1P1,4,5 Receptor Modulator, Exhibits Differential G Protein Signaling Compared to Other S1P Receptor Modulators

BackgroundEtrasimod (APD334) is an oral, selective sphingosine‐1‐phosphate (S1P) receptor modulator in development for atopic dermatitis and inflammatory bowel disease. In humans, S1P receptor modulators decrease heart rate at first‐dose due to initial on‐target S1P receptor 1 (S1P1) agonism in cardiac tissue. S1P1 agonism activates Gi and β‐arrestin signaling pathways: β‐arrestin activation leads to receptor internalization, the proposed mechanism by which S1P receptor modulators regulate lymphocyte trafficking; Gi‐coupled signaling activates G‐protein coupled inwardly rectifying potassium (GIRK) channels, that may contribute to the regulation of heart rate. Approved or investigational S1P receptor modulators have demonstrated different degrees of heart rate reduction and atrioventricular block in clinical studies. Such differences may be the result of differential signaling through the Gi coupled GIRK pathway.AimTo investigate S1P receptor expression in human sinoatrial (SA) node tissue, and the relative S1P1 mediated signaling events and GIRK channel activation of etrasimod and other S1P receptor modulators in the context of first dose heart rate effects.MethodsS1P receptor profiling was performed by qPCR in normal human primary SA node and right atrium samples. Potencies for multiple S1P receptor modulators to activate different S1P1 downstream signaling pathways (GTPγS, cAMP, β‐arrestin, S1P1 internalization) were measured in recombinant human‐S1P1 expressing cells. Gi mediated inhibition of cAMP was also measured in primary HUVEC. GIRK channel activation was measured in isolated adult human primary atrial myocytes.ResultsProfiling of S1P receptors in normal human atrial tissue and SA node demonstrated enriched expression of S1P1,2,3 with only minor expression of S1P4,5. Etrasimod was equipotent to other S1P receptor‐ modulators such as ozanimod, fingolimod, and siponimod in promoting β‐arrestin recruitment and S1P1 receptor internalization. Etrasimod was significantly less potent than ozanimod, fingolimod, and siponimod in assays measuring G‐protein activation (GTPγS binding and cAMP inhibition). In comparisons of GIRK activation, etrasimod was found to be a less potent activator of human atrial GIRK channels than ozanimod.ConclusionsWhen compared to other S1P1 modulators, the pharmacological profile of etrasimod at the S1P1 receptor suggests diminished capacity for signaling through Gi‐protein dependent pathways believed to be responsible for transient reductions in heart rate, while maintaining potent signaling in the β‐arrestin recruitment and receptor internalization related pathways underpinning the therapeutic efficacy of S1P1 modulators in immune‐mediated inflammatory disease settings.Support or Funding InformationThe study was sponsored by Arena Pharmaceuticals, Inc.

Carboxy-Terminal Phosphoregulation of the Long Splice Isoform of Free-Fatty Acid Receptor-4 Mediates β-Arrestin Recruitment and Signaling to ERK1/2

Free-fatty acid receptor-4 (FFA4), previously termed GPR120, is a G protein-coupled receptor (GPCR) for medium and long-chained fatty acids, agonism of which can regulate a myriad of metabolic, sensory, inflammatory, and proliferatory signals. Two alternative splice isoforms of FFA4 exist that differ by the presence of an additional 16 amino acids in the longer (FFA4-L) transcript, which has been suggested to be an intrinsically β-arrestin-biased GPCR. Although the shorter isoform (FFA4-S) has been studied more extensively, very little is known about mechanisms of regulation or signaling of the longer isoform. Because β-arrestin recruitment is dependent on receptor phosphorylation, in the current study, we used the endogenous agonist docosahexaenoic acid (DHA) to examine the mechanisms of FFA4-L phosphorylation, as well as DHA-dependent β-arrestin recruitment and DHA-dependent extracellular-signal regulated kinase-1/2 (ERK1/2) signaling in human embryonic kidney 293 cells. Our results reveal differences in basal phosphorylation of the two FFA4 isoforms, and we show that DHA-mediated phosphorylation of FFA4-L is primarily regulated by G protein-coupled receptor kinase 6, whereas protein kinase-C can also contribute to agonist-induced and heterologous phosphorylation. Moreover, our data demonstrate that FFA4-L phosphorylation occurs on the distal C terminus and is directly responsible for recruitment and interactions with β-arrestin-2. Finally, using CRISPR/Cas9 genome-edited cells, our data reveal that unlike FFA4-S, the longer isoform is unable to facilitate phosphorylation of ERK1/2 in cells that are devoid of β-arrestin-1/2. Together, these results are the first to demonstrate phosphoregulation of FFA4-L as well as the effects of loss of phosphorylation sites on β-arrestin recruitment and ERK1/2 activation. SIGNIFICANCE STATEMENT: Free-fatty acid receptor-4 (FFA4) is a cell-surface G protein-coupled receptor for medium and long-chained fatty acids that can be expressed as distinct short (FFA4-S) or long (FFA4-L) isoforms. Although much is known about FFA4-S, the longer isoform remains virtually unstudied. Here, we reveal the mechanisms of docosahexaenoic acid-induced phosphorylation of FFA4-L and subsequent β-arrestin-2 recruitment and extracellular-signal regulated kinase-1/2 activity.

In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity.

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH24) and hypo-glycosylated FSH (FSH18/21) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH18/21 and FSH24) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH18/21, FSH24, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH18/21 followed by eFSH, recFSH, and FSH24. In β-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH18/21 exhibited a preference toward β-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of β-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH18/21. Concurrent silencing of β-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and β-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca2+ (iCa2+) accumulation through both influx from Ca2+ channels and release from intracellular stores; however, iCa2+ in response to FSH18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa2+ accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.

Morphine‐induced respiratory depression is independent of β‐arrestin2 signalling

Background and PurposeGPCRs can signal through both G proteins and β‐arrestin2. For the μ‐opioid receptor, early experimental evidence from a single study suggested that G protein signalling mediates analgesia, whereas β‐arrestin2 signalling mediates respiratory depression and constipation. Consequently, for more than a decade, much research effort has been focused on developing biased μ‐opioid agonists that preferentially target G protein signalling over β‐arrestin signalling, as it was believed that such drugs would be analgesics devoid of respiratory depressant activity. However, the prototypical compounds that have been developed based on this concept have so far failed in clinical and preclinical development.Experimental ApproachThe present study was set up to re‐examine opioid‐induced respiratory depression in β‐arrestin2 knockout mice. To this end, a consortium was formed consisting of three different laboratories located in different countries to evaluate independently opioid‐induced respiratory depression.Key ResultsOur consensus results unequivocally demonstrate that the prototypical μ‐opioid agonist morphine (3.75–100 mg·kg−1 s.c. or 3–30 mg·kg−1 i.p.) as well as the potent opioid fentanyl (0.05–0.35 mg·kg−1 s.c.) do indeed induce respiratory depression and constipation in β‐arrestin2 knockout mice in a dose‐dependent manner indistinguishable from that observed in wild‐type mice.Conclusion and ImplicationsOur findings do not support the original suggestion that β‐arrestin2 signalling plays a key role in opioid‐induced respiratory depression and call into question the concept of developing G protein‐biased μ‐opioid receptor agonists as a strategy for the development of safer opioid analgesic drugs.