Characterization of Proton Sensitive G protein-Coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) are membrane-bound receptors that can stimulate an intracellular signaling pathway following activation by a ligand. According to the International Union of Basic and Clinical Pharmacology (IUPHAR) database, GPR4, GPR65, and GPR132 are Class A orphan GPCRs with protons reported as their putative endogenous ligand; however, these receptors are currently understudied. After confirming whether these receptors are pH-sensitive, the purpose of our study was to investigate the interactions between GPR4, GPR65 and GPR132 and G protein subtypes (G?s, G?i, G?q, and G?12) upon stimulation with an acidic solution. Using bioluminescence resonance energy transfer (BRET), we studied the coupling between luciferase-tagged GPR receptors and fluorescent protein (Venus)-tagged G proteins in response to pH changes. Data indicated that all three receptors responded to pH changes. Upon extracellular response to pH changes, the receptors activate different G protein subtypes and thus, different signaling pathways: GPR4 activates G?i, G?q, and G?12; GPR65 activates all four subtypes; and GPR132 activates G?i�and weakly activates G?q, and G?12. Identifying these receptors as true proton sensors leads the way in understanding the role they play in maintaining acid-base homeostasis and will be critical for the development of novel drugs combatting acid-base related disorders.