Agonist dependency of the second phase access of ß-arrestin 2 to the heteromeric µ-V1b receptor.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to ß-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that ß-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and ß-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, ß-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of ß-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of ß-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

ß-arrestin 2 as an activator of cGAS-STING signaling and target of viral immune evasion.

Virus infection may induce excessive interferon (IFN) responses that can lead to host tissue injury or even death. ß-arrestin 2 regulates multiple cellular events through the G protein-coupled receptor (GPCR) signaling pathways. Here we demonstrate that ß-arrestin 2 also promotes virus-induced production of IFN-ß and clearance of viruses in macrophages. ß-arrestin 2 interacts with cyclic GMP-AMP synthase (cGAS) and increases the binding of dsDNA to cGAS to enhance cyclic GMP-AMP (cGAMP) production and the downstream stimulator of interferon genes (STING) and innate immune responses. Mechanistically, deacetylation of ß-arrestin 2 at Lys171 facilitates the activation of the cGAS-STING signaling and the production of IFN-ß. In vitro, viral infection induces the degradation of ß-arrestin 2 to facilitate immune evasion, while a ß-blocker, carvedilol, rescues ß-arrestin 2 expression to maintain the antiviral immune response. Our results thus identify a viral immune-evasion pathway via the degradation of ß-arrestin 2, and also hint that carvedilol, approved for treating heart failure, can potentially be repurposed as an antiviral drug candidate.

Identification of psychedelic new psychoactive substances (NPS) showing biased agonism at the 5-HT2AR through simultaneous use of ß-arrestin 2 and miniGαq bioassays.

Psychedelic new psychoactive substances (NPS), compounds exerting their main pharmacological effects through the activation of the serotonin 2A receptor (5-HT2AR), continuously comprise a substantial portion of the reported NPS. However, these substances and their exact mechanism of action, differentiating them from non-psychedelic 5-HT2AR agonists, require further characterization. One potentially relevant phenomenon is the occurrence of biased agonism, in which (a) certain signaling pathway(s) is preferentially activated over the other(s). To this end, a new bioassay was developed, monitoring the recruitment of an engineered miniGαq protein to the activated 5-HT2AR. The setup was designed to be analogous to that of a previously developed bioassay monitoring ß-arrestin 2 recruitment through the NanoBiT system, enabling estimation of the potential preference of a substance to trigger recruitment of one protein over the other. This approach yielded several statistically significantly biased agonists within the group of phenylalkylamine psychedelics, more specifically the N-benzyl substituted 25H analogues 25H-NBF, 25H-NBMD, 25H-NBOH and 25H-NBOMe. All four compounds show a statistically significant preference towards the recruitment of ß-arrestin 2 over miniGαq, as compared to the reference psychedelic substance LSD. We identified markedly different responses for Bromo-DragonFLY in the two bioassays, suggesting biased agonism, though the calculated bias factor equalled out to approximately 0. This demonstrates that the accurate assessment of biased agonism requires both the consideration of the observed trends in addition to the numerical value of the bias factor. A second panel of structural (I-substituted) analogues of the former group of phenylalkylamines showed a similar trend in the ranking order of the bias factors, resulting in one additional compound (25I-NBF) being statistically significantly biased.

A Split Luciferase Complementation Assay for the Quantification of ß-Arrestin2 Recruitment to Dopamine D2-Like Receptors.

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of ß-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify ß-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved ß-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no ß-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the ß-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/ß-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.

Biased versus Partial Agonism in the Search for Safer Opioid Analgesics.

Opioids such as morphine-acting at the mu opioid receptor-are the mainstay for treatment of moderate to severe pain and have good efficacy in these indications. However, these drugs produce a plethora of unwanted adverse effects including respiratory depression, constipation, immune suppression and with prolonged treatment, tolerance, dependence and abuse liability. Studies in ß-arrestin 2 gene knockout (ßarr2(-/-)) animals indicate that morphine analgesia is potentiated while side effects are reduced, suggesting that drugs biased away from arrestin may manifest with a reduced-side-effect profile. However, there is controversy in this area with improvement of morphine-induced constipation and reduced respiratory effects in ßarr2(-/-) mice. Moreover, studies performed with mice genetically engineered with G-protein-biased mu receptors suggested increased sensitivity of these animals to both analgesic actions and side effects of opioid drugs. Several new molecules have been identified as mu receptor G-protein-biased agonists, including oliceridine (TRV130), PZM21 and SR-17018. These compounds have provided preclinical data with apparent support for bias toward G proteins and the genetic premise of effective and safer analgesics. There are clinical data for oliceridine that have been very recently approved for short term intravenous use in hospitals and other controlled settings. While these data are compelling and provide a potential new pathway-based target for drug discovery, a simpler explanation for the behavior of these biased agonists revolves around differences in intrinsic activity. A highly detailed study comparing oliceridine, PZM21 and SR-17018 (among others) in a range of assays showed that these molecules behave as partial agonists. Moreover, there was a correlation between their therapeutic indices and their efficacies, but not their bias factors. If there is amplification of G-protein, but not arrestin pathways, then agonists with reduced efficacy would show high levels of activity at G-protein and low or absent activity at arrestin; offering analgesia with reduced side effects or 'apparent bias'. Overall, the current data suggests-and we support-caution in ascribing biased agonism to reduced-side-effect profiles for mu-agonist analgesics.

Crystal Structure of ß-Arrestin 2 in Complex with CXCR7 Phosphopeptide.

ß-Arrestins (ßarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. ßarrs have two isoforms, ßarr1 and ßarr2. Receptor phosphorylation is a key determinant for the binding of ßarrs, and understanding the intricate details of receptor-ßarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active ßarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of ßarr2 remains elusive. Here, we present a 2.3-Å crystal structure of ßarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound ßarr2 reveals key differences from the previously determined active conformation of ßarr1. One of the key differences is that C7pp-bound ßarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of ßarr2 and suggested that ßarr2 adopts a range of inter-domain rotations.

Carvedilol Diminishes Cardiac Remodeling Induced by High-Fructose/High-Fat Diet in Mice via Enhancing Cardiac ß-Arrestin2 Signaling.

BACKGROUND: Insulin resistance (IR) is a well-known risk factor for cardiovascular complications. This study aimed to investigate the effect of a dietary model of IR in mice on cardiac remodeling, cardiac ß-arrestin2 signaling, and the protective effects of carvedilol as a ß-arrestin-biased agonist. METHODS AND RESULTS: Insulin resistance was induced by feeding mice high-fructose/high-fat diet (HFrHFD) for 16 weeks. Carvedilol was adiministered for 4 weeks starting at week 13. At the end of the experiment, body weight, heart weight, left and right ventricular thickness, visceral fat weight, fasting blood glucose (FBG), serum insulin, IR index, and serum endothelin-1 were measured. In addition, cardiac tissue samples were histopathologically examined. Also, cardiac levels of cardiotrophin-1, ß-arrestin2, phosphatidylinositol 4,5 bisphosphate (PIP2), diacylglycerol (DAG), and phosphoserine 473 Akt (pS473 Akt) were measured. Results showed significant increases in the FBG, serum insulin, IR index, serum endothelin-1, cardiac DAG, cardiac fibrosis, and degenerated cardiac myofibrils in HFrHFD-fed mice associated with a significant reduction in cardiac levels of cardiotrophin-1, ß-arrestin2, PIP2, and pS473 Akt. On the other hand, carvedilol significantly reduced the heart weight, FBG, serum insulin, IR index, serum endothelin-1, cardiac DAG, left ventricular thickness, right ventricular fibrosis, and degeneration of cardiac myofibrils. In addition, carvedilol significantly increased cardiac levels of cardiotrophin-1, ß-arrestin2, PIP2, and pS473 Akt. CONCLUSION: Carvedilol enhances cardiac ß-arrestin2 signaling and reduces cardiac remodeling in HFrHFD-fed mice.

Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells.

Glioblastoma is among the most aggressive brain tumors and has an exceedingly poor prognosis. Recently, the importance of the tumor microenvironment in glioblastoma cell growth and progression has been emphasized. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and endogenous ligands originating from dying cells or the extracellular matrix involved in host defense and in inflammation. G-protein coupled receptors (GPCRs) have gained interest in anti-tumor drug discovery due to the role that they directly or indirectly play by transactivating other receptors, causing cell migration and proliferation. A proteomic analysis showed that the nociceptin receptor (NOPr) is among the GPCRs significantly expressed in glioblastoma cells, including U87 cells. We describe a novel role of the peptide nociceptin (N/OFQ), the endogenous ligand of the NOPr that counteracts cell migration, proliferation and increase in IL-1ß mRNA elicited by LPS via TLR4 in U87 glioblastoma cells. Signaling pathways through which N/OFQ inhibits LPS-mediated cell migration and elevation of [Ca2+]i require ß-arrestin 2 and are sensitive to TNFR-associated factor 6, c-Src and protein kinase C (PKC). LPS-induced cell proliferation and increase in IL-1ß mRNA are counteracted by N/OFQ via ß-arrestin 2, PKC and extracellular signal-regulated kinase 1/2; furthermore, the contributions of the transcription factors NF-kB and AP-1 were investigated. Independent of LPS, N/OFQ induces a significant increase in cell apoptosis. Contrary to what was observed in other cell models, a prolonged exposure to this endotoxin did not promote any tolerance of the cellular effects above described, including NOPr down-regulation while N/OFQ loses its inhibitory role.

ß-Arrestin-biased AT1R stimulation promotes extracellular matrix synthesis in renal fibrosis.

The renin-angiotensin system plays a critical role in the progression of renal fibrosis. Angiotensin II type 1 receptor (AT1R) belongs to the B family of the G protein-coupled receptor (GPCR) family. ß-Arrestins are known as negative regulators of GPCRs. Recently, ß-arrestins have been found to regulate multiple intracellular signaling pathways independent of G proteins. In this study we investigated the role of ß-arrestins in regulating extracellular matrix (ECM) synthesis in renal fibrosis. The rat kidney fibroblast cell line (NRK-49F) was treated with the ß-arrestin biased agonist [1-sar, 4, 8-ile]angiotensin II (SII), which does not initiate AT1R-G protein signaling. The cells were transfected with recombinant adenoviruses expressing ß-arrestin-2 gene or small-interfering RNA (siRNA) targeting ß-arrestin-2. The unilateral ureteral obstruction (UUO) model was used in vivo. mRNA and protein levels of ß-arrestin-2, not ß-arrestin-1, were significantly upregulated in the UUO kidney tissues. SII induced the tight binding of ß-arrestin-2 with AT1R. SII increased the synthesis of collagen I and fibronectin in NRK-49F, which were abolished when pretreated with candesartan (AT1R blocker). Transfection of siRNA targeting ß-arrestin-2 decreased the effects of SII on ECM synthesis. Overexpression of ß-arrestin-2 enhanced SII-stimulated ECM synthesis. SII induced ERK1/2 phosphorylation in NRK-49F. Transfection of siRNA targeting ß-arrestin-2 inhibited ERK phosphorylation. Overexpression of ß-arrestin-2 increased ERK1/2 phosphorylation. Our study first showed that AT1R-ß-arrestin-2 pathway signaling plays an important role in renal fibrosis, although it was previously believed that the AT1R-G protein pathway plays a major role. Targeting ß-arrestin-2 may be a potential therapeutic agent for renal fibrosis.

Activation of the Orphan G Protein-Coupled Receptor GPR27 by Surrogate Ligands Promotes ß-Arrestin 2 Recruitment.

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in ß-arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of ß-arrestin 2 to a GPR27V2 chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [N-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro-N-{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a N-phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology ß-arrestin 2 assay, imaging of green fluorescent protein-tagged ß-arrestin 2, and PathHunter ß-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.

Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands.

BACKGROUND AND PURPOSE: Nociceptin/orphanin FQ (N/OFQ) receptor (NOP) agonists produce anxiolytic-like effects in rodents while antagonists promote antidepressant-like effects. The aim of this study was to investigate the effect on anxiety and depression of NOP receptor partial agonists such as the peptides [F/G]N/OFQ(1-13)NH2 and UFP-113 and the non-peptide AT-090. EXPERIMENTAL APPROACH: In vitro AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 were tested for their ability to promote NOP/G-protein and NOP/ß-arrestin 2 interaction, using a bioluminescence resonance energy transfer assay. In vivo, they were tested in mice in the elevated plus maze (EPM) and in the forced swim (FST) tests. NOP partial agonists effects were systematically compared to those of full agonists (N/OFQ and Ro 65-6570) and antagonists (UFP-101 and SB-612111). KEY RESULTS: In vitro, AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 promoted NOP/G protein interaction, with maximal effects lower than those evoked by N/OFQ and Ro 65-6570. AT-090 behaved as a NOP partial agonist also in inducing ß-arrestin 2 recruitment, while UFP-113 and [F/G]N/OFQ(1-13)NH2 were inactive in this assay. In vivo, AT-090 induced anxiolytic-like effects in the EPM but was inactive in the FST. Opposite results were obtained with UFP-113 and [F/G]N/OFQ(1-13)NH2. CONCLUSIONS AND IMPLICATIONS: NOP ligands producing similar effects on NOP/G protein interaction (partial agonism) but showing different effects on ß-arrestin 2 recruitment (partial agonism vs antagonism) elicited different actions on anxiety and mood. These results suggest that the action of a NOP ligand on emotional states is better predicted based on its ß-arrestin 2 rather than G-protein efficacy.