British journal of pharmacology · 2026
The chemokine receptor-like fourth extracellular loop of the apelin receptor differentially regulates apelin and elabela binding and signalling.
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Bonef Chloé, Bouhsine Hela, Huynh Carine, Chaigneau Marion, Dubois Steven, Garcia-Marcos Mikel, Llorens-Cortes Catherine, Servent Denis, Robin Philippe, Nozach Hervé, Alves Isabel, Iturrioz Xavier
Аннотация
BACKGROUND AND PURPOSE: Apelin 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 RESULTS: Disruption 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.
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 RESULTS: Disruption 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.