Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.

Ceramides play a central role in human health and disease, yet their role as systemic signaling molecules remain poorly understood. In this work, we identify formyl peptide receptor 2 (FPR2) as a membrane receptor that specifically binds long-chain ceramides (C14 to C20). In brown and beige adipocytes, C16:0 ceramide binding to FPR2 inhibits thermogenesis through G _i cyclic adenosine monophosphate signaling pathways, an effect that is reversed in the absence of FPR2. We present three cryo–electron microscopy structures of FPR2 in complex with G _i trimers bound to C16:0, C18:0, and C20:0 ceramides. The hydrophobic tails are deeply embedded in the orthosteric ligand pocket, which has a limited amount of plasticity. Modification of the ceramide binding motif in closely related receptors, such as FPR1 or FPR3, converts them from inactive to active ceramide receptors. Our findings provide a structural basis for adipocyte thermogenesis mediated by FPR2.

Background and Purpose: Cholecystokinin (CCK) is secreted by intestinal I cells and regulates important metabolic functions. In pancreatic islets, CCK controls beta cell functions primarily through CCK₁receptors, but the signalling pathways downstream of these receptors in pancreatic beta cells are not well defined.Experimental Approach: Apoptosis in pancreatic beta cell apoptosis was evaluated using Hoechst‐33342 staining, TUNEL assays and Annexin‐V‐FITC/PI staining. Insulin secretion and second messenger production were monitored using ELISAs. Protein and phospho‐protein levels were determined by Western blotting. A glucose tolerance test was carried out to examine the functions of CCK‐8s in streptozotocin‐induced diabetic mice.Key Results: The sulfated carboxy‐terminal octapeptide CCK_26‐33amide (CCK‐8s) activated CCK₁receptors and induced accumulation of both IP₃and cAMP. Whereas G_q‐PLC‐IP₃signalling was required for the CCK‐8s‐induced insulin secretion under low‐glucose conditions, G_s‐PKA/Epac signalling contributed more strongly to the CCK‐8s‐mediated insulin secretion in high‐glucose conditions. CCK‐8s also promoted formation of the CCK₁receptor/β‐arrestin‐1 complex in pancreatic beta cells. Using β‐arrestin‐1 knockout mice, we demonstrated that β‐arrestin‐1 is a key mediator of both CCK‐8s‐mediated insulin secretion and of its the protective effect against apoptosis in pancreatic beta cells. The anti‐apoptotic effects of β‐arrestin‐1 occurred through cytoplasmic late‐phase ERK activation, which activates the 90‐kDa ribosomal S6 kinase‐phospho–Bcl‐2‐family protein pathway.Conclusions and Implications: Knowledge of different CCK₁receptor‐activated downstream signalling pathways in the regulation of distinct functions of pancreatic beta cells could be used to identify biased CCK₁receptor ligands for the development of new anti‐diabetic drugs.

The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part because no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W^6.53 and residues of the ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15-induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2 but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.