Probing Membrane Catalyzed Apelin-Receptor Interactions by Fluorescence Spectroscopy

Abstract

Apelin is a peptidic hormone that activates the class A G-protein-coupled apelin receptor. Apelin is found in several bioactive isoforms in the body, ranging from 13 to 55 amino acids in length. Only the C-terminal 12 amino acids are required for bioactivity. Apelin-17 has previously been shown to bind to micelles of anionic detergents, undergoing a membrane-induced structural transition. It has been theorized that a peptide-membrane interaction and the resulting conformational changes are necessary for peptide recognition by the receptor, while also serving to increase local concentration of the ligand in what is known as the membrane catalysis theory. Here, a method is described for conjugating both synthetic and recombinant apelin to fluorescent probes. These fluorescently-labelled peptides were then used in fluorescence experiments to test for membrane-interaction, as required by the membrane catalysis theory. It was found that all three isoforms of apelin interact favourably with both anionic and zwitterionic detergents, as indicated by the calculated Py values. Förster resonance energy transfer (FRET) experiments were performed with fluorescently-labelled apelin-17 to native tryptophan in a fragment of the apelin receptor to both localize the ligand-receptor binding interface and to quantify the peptide-receptor interaction.