Apelin binding to human APJ receptor leads to biased signaling

Human 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.

Based 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.

Stimulation 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 G_i-biased signaling while ²⁶⁸KTL²⁷⁰-AAA in ECL3 showed G_q biasing. C281A mutant in ECL3 was deficient in all assays.

Conserved 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.

This work explains the importance of extracellular loop domains in ligand binding, receptor activation and downstream signaling of human APJR.