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The human APJ receptor (APJR), activated by apelin isoforms, regulates cardiovascular functions and fluid homeostasis. Understanding its structure‐function relationship is crucial for a comprehensive knowledge of signalling aberrations that cause several physiological disorders. Here, we demonstrate the influence of extracellular loop (ECL) domains in the mechanism of β‐arrestin‐mediated signalling from human APJR: Apelin system. Alanine mutations of evolutionarily conserved residues were characterized using receptor internalization, β‐arrestin pull‐down, Akt phosphorylation and cell migration assay. C281A and 268KTL270‐AAA in ECL3 were deficient in all assays, whereas 183MDYS186‐AAAA mutant in ECL2 showed impaired β‐arrestin‐mediated signalling but demonstrated Gi‐dependent cell migration. Our findings establish that conserved residues in the extracellular domain play a prominent role in modulating receptor interactions with the β‐arrestin signalling cascade.

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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 Gq‐, Gi‐, and β‐arrestin‐mediated signaling. Mutation‐dependent variation in ligand binding and signaling was observed. W197A in ECL2 and L276L277W279‐AAA in ECL3 were deficient in Gi and β‐arrestin signaling pathways with relatively preserved Gq‐mediated signaling. T169T170‐AA, Y182A, and T190A mutants in ECL2 showed impaired β‐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.

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