Probing GPCR Activation: Functional Analysis of the N-terminus and Extracellular Loops of the Apelin Receptor

Abstract

The apelin receptor (AR or APJ) is a class A (rhodopsin-like) G-protein coupled receptor (GPCR) with wide distribution throughout the human body. Activation of AR by its cognate peptidic ligand, apelin, induces diverse physiological effects including vasoconstriction and dilation; strengthening of heart muscle contractility; angiogenesis; and, regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish embryonic development. The AR is also implicated in several pathologies, including cardiovascular disease, diabetes, obesity and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about the mechanism by which peptides activate the AR. Additional complexity arises because the AR is modulated by two endogenous peptide ligands, both of which appear to have multiple bioactive isoforms of varying length. This is compounded by the fact that the various apelin and Toddler/ELABELA isoforms are differently distributed and produce distinct cellular effects. This work aims to identify functionally critical amino acid residues on the extracellular face of the AR, as this region may contribute to the specificity and affinity of apelin binding, and to larger conformational changes preceding AR activation and internalization. Alanine-substitution mutants of negatively charged and aromatic residues in the N-terminus and extracellular loops 1 and 2 of the AR were prepared. Apelin-13-induced activation of the wild-type (WT) AR and mutants was then evaluated in HEK293A cells using western blotting to measure extracellular-signal-regulated kinase (ERK) activation. These studies revealed several mutations of interest. In particular two N-terminal mutants, E20A and D23A, previously shown to have reduced apelin-binding affinity and internalization, exhibit maximal apelin-induced ERK activation comparable to the WT AR. This suggests that the binding affinity and ability of apelin to activate downstream signalling via the AR are two separate parameters. Lastly, one element of this work was the production and purification of recombinant apelin-36, to be used in future structural and functional studies of AR ligand-binding and activation.