Elabela/Toddler and apelin bind differently to the apelin receptor.

Like apelin (pE13F, K17F), Elabela/Toddler is an endogenous ligand of the apelin receptor playing a key role in cardiovascular development. Elabela/Toddler exists as peptide fragments of 32 (Q32P), 22 (K22P) and 11 (C11P) amino acids. In this study, we investigated the possible structural and functional similarities between these endogenous ligands. We performed in vitro pharmacological characterization and biased signaling analyses for apelin and Elabela/Toddler fragments in CHO cells, by assessing binding affinities, the inhibition of cyclic adenosine monophosphate (cAMP) production and the triggering of ß-arrestin 2 recruitment. We also performed Alanine scanning for Elabela/Toddler and structure-function studies based on site-directed mutagenesis of the rat and human apelin receptor, to compare the modes of binding of the different endogenous ligands. Alanine scanning of K22P showed that neither of its cysteine residues were involved in binding or in peptide activity and that its C-terminus carried the key pharmacophore for receptor binding and activation. We showed that Asp282 and Asp284 of rat and human apelin receptor, respectively, were not involved in Elabela/Toddler activity, whereas they are key residues for apelin binding and activity. We found that the structural features of Elabela/Toddler and apelin were different, resulting in different modes of binding of these endogenous ligands to the apelin receptor. These differences should be taken into account in the future development metabolically stable analogs of Elabela/Toddler and apelin as potential therapeutic tools for the treatment of cardiovascular diseases and water retention/hyponatremic disorders.