Dissecting the Roles of the Extracellular Regions in Apelin Peptide-Receptor Binding

Abstract

The apelin receptor is a class A G-protein coupled receptor with homology to the angiotensin II receptor and the CXCR4 and CCR5 chemokine receptors. It is expressed at high levels in lung, heart, adipose tissue, kidney, spleen and in some areas of the brain. The apelin receptor gene is also upregulated and overexpressed in one-third of human tumours. The receptor is assumed to consist of a transmembrane region with 7 helical segments, an intracellular and an extracellular domain. The extracellular domain, made up of the N-terminal tail and three extracellular loops (EL1, 2 and 3), exhibits low sequence homology to other GPCRs. Several somatic mutations in loops have been linked to diseases, pointing out the importance of loops in the activation and/or binding of GPCRs to their cognate ligands. We carried out a physicochemical characterization of the N-terminus and extracellular loops of the apelin receptor in lipid mimetic environments. The N-terminus (attached to the first transmembrane domain) and EL2 were produced as recombinant proteins while the EL peptides and a fluorescently tagged apelin were synthesized by solid-phase peptide synthesis. All species were purified by high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between apelin and extracellular loop peptides was carried out using circular dichroism spectroscopy, fluorescence resonance energy transfer and nuclear magnetic resonance spectroscopy. These results provide insight into understanding the role of the each of the extracellular regions of this receptor at the molecular level and constitute important information the apelin receptor for the development of therapeutics targeting this system.