The chemokine receptor-like fourth extracellular loop of the apelin receptor differentially regulates apelin and elabela binding and signalling.

background and purposeApelin and Elabela are endogenous ligands of the apelin receptor (apelin receptor/APJ), a GPCR involved in cardiovascular regulation and body fluid homeostasis. The human receptor contains a conserved disulfide bridge linking the N-terminal domain to extracellular loop 3 (ECL3) via Cys19 and Cys281, forming a structural constraint analogous to a fourth extracellular loop ('ECL4'). In the chemokine receptor family, this motif plays a critical role in ligand engagement and receptor activation. EXPERIMENTAL APPROACH: The functional role of this disulfide bridge was investigated using site-directed mutagenesis (Cys19Ala and Cys281Ala), plasmon waveguide resonance binding assays and BRET-based biosensors to monitor Gαi activation and β-arrestin-2 recruitment. KEY

resultsDisruption of the disulfide bridge did not affect receptor surface expression but markedly impaired binding of apelin fragments (pE13F and K17F), leading to reduced Gαi signalling and β-arrestin-2 recruitment. In contrast, binding of the Elabela fragment K22P and the subsequent apelin receptor signalling activation were largely preserved. Antagonist antibodies (JN241 and JN241-Fc) retained activity, whereas the agonist antibody JN241-9-Fc showed strongly reduced efficacy for the mutated receptors. CONCLUSION AND IMPLICATIONS: The N-terminal/ECL3 disulfide bridge is a critical structural determinant for apelin binding and apelin receptor activation but is less critical for Elabela signalling, supporting distinct modes of ligand engagement. Antibody-mediated agonism is particularly sensitive to alterations of « ECL4 » without affecting its binding, suggesting that this structural constraint is required for optimal apelin receptor activation. The apelin receptor shares functional similarities with the chemokine receptors with respect to this.