Apelin binding to human APJ receptor leads to biased signaling

•Conserved residues across various species of APJR were mutated•Mutation dependent variation in intracellular Ca2+ rise, receptor internalization and ERK1/2 phosphorylation was observed•183MDYS186 -AAAA in ECL2 showed Gi biased activation•C281 in ECL3 might be involved in a disulfide linkage important for receptor activation

BackgroundHuman APJ receptor (APJR), a rhodopsin family G-Protein Coupled Receptor (GPCR), activated by isoforms of peptide ligand apelin causing potent inotropic effect, is involved in cardiac function, angiogenesis and maintenance of fluid homeostasis. APJR is expressed in various organs e.g., heart, brain, kidney, muscles, etc. Hence, problems in APJR signaling lead to severe dysregulation in the pathophysiology of an organism.MethodsBased on multiple sequence alignment of receptors from various organisms, we observe a large number of conserved residues in the extracellular side. Mutational studies including calcium mobilization, receptor internalization and ERK1/2 phosphorylation assays were performed.ResultsStimulation of APJR and its mutants with apelin-13 led to mutation-dependent variation in receptor activation, intracellular Ca2+ rise, and its subsequent downstream signaling. The mutant 183MDYS186-AAAA in ECL2 showed Gi-biased signaling while 268KTL270-AAA in ECL3 showed Gq biasing. C281A mutant in ECL3 was deficient in all assays.ConclusionConserved residues in the ECL2 of APJR are key for ligand binding, activation mechanism, and selective downstream signaling. Additionally, we demonstrate that Cys281 (in ECL3) mediated disulfide linkage is important for ligand recognition and receptor activation.General significanceThis work explains the importance of extracellular loop domains in ligand binding, receptor activation and downstream signaling of human APJR.