Critical APJ receptor residues in extracellular domains that influence effector selectivity

Human APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and G(q)-, G(i)-, and beta-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W(197)A in ECL2 and (LLW279)-L-276-W-277-AAA in ECL3 were deficient in G(i) and beta-arrestin signaling pathways with relatively preserved G(q)-mediated signaling. (TT170)-T-169-AA, Y(182)A, and T(190)A mutants in ECL2 showed impaired beta-arrestin-dependent cell signaling while maintaining G protein(-)mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions.