G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.

Novel findings reveal important functional roles for β-arrestin 1 and β-arrestin 2 in the regulation of insulin secretion, β-cell survival, and β-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 knock down β-arrestins by small interference RNA, to follow subcellular localization of β-arrestins in the cytosol and nucleus of the insulinoma INS-1E rat pancreatic β-cell line, and to analyze β-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 β-arrestin 2 knockout (Arrb2-/-) mice and to evaluate β-arrestin-mediated roles in β-cell mass plasticity and β-cell signaling using immunocytochemistry on pancreatic sections or on primary dispersed β-cells from wild-type mice and Arrb2-/- mice.

A limited number of biomarkers in the central and peripheral systems which are known may be useful for diagnosing major depressive disorders and predicting the effectiveness of antidepressant (AD) treatments. Since 60% of depressed patients do not respond adequately to medication or are resistant to ADs, it is imperative to delineate more accurate biomarkers. Recent clinical studies suggest that β-arrestin 1 levels in human mononuclear leukocytes may be an efficient biomarker. If potential biomarkers such as β-arrestin 1 could be assessed from a source such as peripheral blood cells, then they could be easily monitored and used to predict therapeutic responses. However, no previous studies have measured β-arrestin 1 levels in peripheral blood mononuclear cells (PBMCs) in anxious/depressive rodents. This study aimed to develop a method to detect β-arrestin protein levels through immunoblot analyses of mouse PBMCs isolated from whole blood. In order to validate the approach, β-arrestin levels were then compared in na\"{\i}ve, anxious/depressed mice, and anxious/depressed mice treated with a selective serotonin reuptake inhibitor (fluoxetine, 18~mg/kg/day in the drinking water). The results demonstrated that mouse whole blood collected by submandibular bleeding permitted isolation of enough PBMCs to assess circulating proteins such as β-arrestin 1. β-Arrestin 1 levels were successfully measured in healthy human subject and na\"{\i}ve mouse PBMCs. Interestingly, PBMCs from anxious/depressed mice showed significantly reduced β-arrestin 1 levels. These decreased β-arrestin 1 expression levels were restored to normal levels with chronic fluoxetine treatment. The results suggest that isolation of PBMCs from mice by submandibular bleeding is a useful technique to screen putative biomarkers of the pathophysiology of mood disorders and the response to ADs. In addition, these results confirm that β-arrestin 1 is a potential biomarker for depression.

Glial cell line-derived neurotrophic factor (GDNF), essential for neuronal survival, plasticity and development, has been implicated in the mechanism of action of antidepressant drugs (ADs). β-arrestin1, a member of the arrestin protein family, was found to play a role in AD mechanism of action. The present study aimed at evaluating whether the effect of ADs on GDNF in C6 rat glioma cells is exerted through a β-arrestin1-dependent, CREB-interactive pathway. For chronic treatment, C6 rat glioma cells were treated for 3 d with different classes of ADs: imipramine - a non-selective monoamine reuptake inhibitor, citalopram - a serotonin selective reuptake inhibitor (SSRI) or desipramine - a norepinephrine selective reuptake inhibitor (NSRI) and compared to mood stabilizers (lithium and valproic acid) or to the antipsychotic haloperidol. Only ADs significantly elevated β-arrestin1 levels in the cytosol, while reducing phospho-β-arrestin1 levels in the cell nuclear fraction. ADs significantly increased both GDNF expression and release from the cells, but were unable to induce such effects in β-arrestin1 knock-down cells. Chronic AD treatment significantly increased CREB phosphorylation without altering the level of total CREB in the nuclear fraction of the cells. Moreover, treatment with ADs significantly increased β-arrestin1/CREB interaction. These findings support the involvement of β-arrestin1 in the mechanism of action of ADs. We suggest that following AD treatment, β-arrestin1 generates a transcription complex involving CREB essential for GDNF expression and release, thus enhancing GDNF's neuroprotective action that promotes cellular survival and plasticity when the survival and function of neurons is compromised as occurs in major depression.

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and β-arrestin at the plasma membrane, and the SP-NK(1)R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling.