The apelinergic system comprises a class A G-protein coupled receptor (GPCR), the apelin receptor (AR; formerly APJ), and two peptidic ligands, apelin and apela. Both ligands are processed into multiple bioactive isoforms in vivo, ranging from ~11 to 55 residues in length. There are also indications that both ligands may bind to alternative, as yet unidentified, GPCR(s). Using solution-state NMR spectroscopy, we have compared and contrasted the interactions of both apelin and apela isoforms with membrane-mimetic micelles and, in the case of apelin, bicelles. The C-terminal region of apelin retained in all bioactive isoforms binds tightly to and becomes structured in the presence of anionic micelles; it also binds to zwitterionic micelles, but structuring is not induced and the peptide remains much more dynamic. Like apelin, apela binds to both zwitterionic and anionic micelles. Segments of helicity are induced, with differences in structuring and binding as a function of isoform length. These observations are consistent with a membrane-catalyzed peptide-receptor interaction. In membrane catalysis, initial nonspecific membrane binding by a peptide ligand increases the likelihood of receptor encounter, interaction, and recognition. Membrane binding may also have ramifications in the proteolytic processing of both apelin and apela to shorter bioactive isoforms, given the membrane-associated nature of many proprotein convertases. To study AR, we have initially been applying the “divide-and-conquer” strategy to facilitate protein production; to characterize AR structure and dynamics; and, to carry out direct structure-function correlation for the AR in advance of production of a full-length protein amenable to NMR spectroscopic study. Through heteronuclear NMR methods, we have compared the behaviour of AR segments in disparate membrane-mimetic environments. AR segments have also served as a model system to compare 19F NMR spectroscopic behaviour of four biosynthetically-incorporated fluorine-substituted tryptophans. This is, in turn, facilitating characterization of ligand-AR interactions and improved throughput of screening for optimal membrane-mimetic conditions for high-resolution NMR study. In the longer-term, comprehensive application of heteronuclear and 19F NMR methods is targeted to develop a mechanistic understanding of all of the key apelinergic system components and their interactions in the membrane environment.

Probing membrane catalysis and ligand-receptor interactions in the apelinergic system

254th National Meeting (2017)

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