The Membrane Catalysis Model: Apelin and its Receptor

Abstract

Apelin is a peptide hormone that activates the class A G-protein coupled apelin receptor. Apelin is found in several bioactive isoforms in the body, ranging from 12 to 55 amino acids in length. Apelin-17 has previously been shown to bind to micelles of anionic detergents, which implies that apelin may interact with cell membranes prior to receptor activation. It has been theorized that this interaction with the membrane may induce conformational changes necessary for peptide recognition by the receptor, while increasing local concentration of the ligand in what is known as the membrane catalysis hypothesis. Here, we describe a method for conjugating various isoforms of apelin to fluorophores, while still maintaining bioactivity, as demonstrated by a phosphorylated extracellular signal-regulated kinase (pERK) assay in apelin receptor-transfected human embryonic kidney cells. These labeled peptides were then used for both fluorescence and diffusion-ordered nuclear magnetic resonance spectroscopy experiments in the presence of varying concentrations of micelle and bicelle species to test for membrane interaction as required by the membrane catalysis model. Forster resonance energy transfer experiments were performed with fluorescently labeled apelin isoforms to native tryptophan in fragments of the apelin receptor and to eGFP-tagged full-length apelin receptor to both localize the ligand-receptor binding interface and to quantify the peptide-receptor interaction.