Molecular determinants on extracellular loop domains that dictate interaction between β‐arrestin and human APJ receptor

Abstract

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.

Critical APJ receptor residues in extracellular domains that influence effector selectivity

“…Receptor internalization assay was done as per our previous report [24]. HEK293T cells, transiently transfected with APJR and its ECL mutant plasmids, were treated with 10 µ m apelin‐13 peptide and incubated further at 37 °C with 5% CO 2 for 1 h. The cells were then fixed on glass slides and receptor internalization was imaged using confocal microscope (Leica DMi8; Leica Microsystems, Wetzlar, Germany), by monitoring the fluorescence signal from eGFP tag.…”

“…Thus, we have identified 'hot spots' in ECL2 (T 169 , T 170 , Y 182 , T 190 , W 197 ) and ECL3 (L 276 , L 277 , W 279 ) of human APJR that control receptor activation by the coordinated movement of extracellular domains, which later transmit to the intracellular domains for effector coupling. Our earlier studies revealed that motifs 183 MDYS 186 and 268 KTL 270 in ECL2 and ECL3 are imperative for biased signaling of apelin receptor [23,24]. In general, hydrophobic residues of ECL2 are presented as important structural motifs that 'lock' the ligand in its binding pocket, and thereby enhance ligand residence time for GPCR interaction [75].…”

“…Radiolabeling of synthetic Apelin peptide (purchased from GenScript, USA) was performed as described earlier [24]. Apelin peptide was briefly incubated with radioactive iodine (Na 125 I) and Chloramine-T.…”

“…Ligand-binding experiments of APJR and its ECL mutants were done as described earlier [24]. Briefly, membrane preparations were prepared from HEK293T cells expressing APJ receptor and its mutants using high-speed centrifugation (100 000 g for 1 h at 4 °C) and stored at -80 °C until further studies.…”

“…In a continuing effort, we aim to address the roles of specific extracellular residues of human APJ receptor involved in not only ligand (apelin peptide) binding/ recognition and mechanism of receptor activation but also preferential downstream signal transduction (G protein and/or β-arrestin) [23,24]. Here, we report the ligand-binding mode of wild-type and mutant receptors using radioactive ligand saturation assays.…”