Functional Characterization Of Neuropeptide Receptor GPR103-Mediated Signaling

Neuropeptides are intercellular signaling molecules secreted by neurons that act as neurotransmitters,neurohormones and neuromodulators to exert a large array of biological activities through different developmental stages.Pyroglutamine RF-amide peptides(QRFPs)are the latest member of the RF-amide peptide family discovered.QRFPs exert their physiological functions by interacting with a specific membrane receptor,G protein-coupled receptor 103(GPR103).GPR103 and its ligands are widely expressed in the brain and peripheral tissues and play important roles in the regulation of a wide range of biological activities such as feeding behavior,energy homeostasis,neuroendocrine function,and bone formation.Up to now,studies suggest potential for the QRFP/GPR103 signaling system as a therapeutic target in the treatment of energy homeostasis and neuroencrine-related diseases.Two mature forms,a 26–amino acid RFamide peptide(26RFa)and its N-extended form,a 43–amino acid peptide(43RFa)have been identified in humans and rodents.Previous studies have reported that both26RFa and 43RFa neuropeptides can specifically bind and activate GPR103,but both26RFa-and 43RFa-mediated signaling pathways remain controversial.In this study,we first used functional assays to examine signaling pathways mediated by 26RFa and43RFa in HEK293T cells.Our results showed that upon stimulation with 26RFa,GPR103 activated G?q and G?i proteins,leading to the inhibition of forskolin-induced c AMP formation,intracellular Ca²⁺mobilization and ERK1/2 phosphorylation,whereas GPR103 coupled to G?q and G?s proteins,resulting in a significant increase in intracellular c AMP production,Ca²⁺mobilization and ERK1/2 phosphorylation in response to treatment of 43RFa.Using the X-ray structure of human endothelin type B receptor-based GPR103 3-D molecular model,we performed molecular dynamics simulations to predict the intermolecular interaction of GPR103 with 43RFa.The docking analysis revealed that Lys¹⁸⁹,Asp¹⁹¹,and Leu²⁰² residues in the ECL2 are critical for the interaction of GPR103 with its ligands 43RFa and 26RFa.The results derived from the combination of site-directed mutagenesis and functional assays showed that upon stimulation with 43RFa peptide,substitution of residue Lys¹⁸⁹ with alanine resulted in the coupling of GPR103 to G?i protein,and replacement of residue Asp¹⁹¹ with alanine led to the interaction of GPR103 with G?q protein,while mutant L202A exhibited switch from G?s and G?q dual coupling to G?i and G?q dual coupling.Moreover,truncation of eight residues from the N-terminus(35RFa)showed the potential to activate G?s and G?q dual-dependent signaling similar to the wild-type43RFa,however,mutant 35RFa-F10A exhibited the change from G?s and G?q dual-dependent signaling to G?i and G?q dual-dependent signaling.In addition,our experiments with the pancreatic islets isolated from mice indicated that 43RFa exhibited higher potential to promote insulin secretion through G?s/c AMP and G?q/Ca²⁺signaling pathways than that of 26RFa,whereas 26RFa also showed the potential to stimulate insulin secretion through G?q/Ca²⁺signaling pathways.Glycosylation is an important process of post-translational modification of GPCR proteins,affecting protein folding,secretion,surface expression,protein localization,transport,ligand binding and other physiological activities.The human QRFP receptor GPR103 possesses three N-glycosylation consensus sites,two located on the N-terminal domain(N5 and N19)and one on the first extracellular loop(ECL1)(N106),however,to date,their role in GPR103 expression and signaling has not been established.Here,we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of GPR103 cell surface expression and signaling.Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1,but not N5 in the N-terminus,served as sites for N-glycosylation.Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of GPR103 is not essentially required for targeting to the cell membrane.However,further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with 26RFa and downstream signaling.Thus,our findings suggest that for the human QRFP receptor GPR103,N-glycosylation is not important for cell surface expression but is a prerequisite for ligand binding and receptor activation.In conclusion,the current studies identify key mechanisms involved in the interaction of GPR103 with its ligands 43RFa and 26RFa and with G proteins.We also showed that N-glycosylation is not important for GPR103 cell surface expression,however,full N-glycosylation is critical for ligand binding and G protein-dependent signaling.Overall,our findings provide novel insights into the agonist-mediated activation of GPR103 and other Class A GPCRs and will support future efforts aimed at better understanding of GPR103 signaling and the discovery of agonist and antagonist drugs targeting GPR103.