Abstract
Living organisms recognize the chemical circumstance through G-protein coupled odorant receptors (ORs), which consist of seven transmembranes (TMs) connected by three intracellular loops (ICLs) and three extracellular loops (ECLs). Although ECLs are the first gateway for chemicals to enter the orthosteric ligand binding site, their structurefunction studies remain incomplete. Among them, ECL3 has been reported to function in the ligand entrance process as a vestibule site in some non-olfactory class A GPCRs and class I odorant receptors. In this study, we sought to determine whether this function of ECL3 can be generalized to all ORs despite the high sequence diversity. To demonstrate this, we performed site-directed mutations of the PxSxxS motif common to class II OR ECL3 for hOR1A1, a prototypical class II OR. In vitro functional assay results revealed that the mutants differentially impact agonists' properties in potency and efficacy. Furthermore, the 3D molecular dynamics also supported that ECL3 of hOR1A1 may function as a vestibule site too. Therefore, this study reconciles the function of ECL3 in ligand selectivity to overall class A GPCR.