Ala-scan of ghrelin (1–14): interaction with the recombinant human ghrelin receptor

Abstract

Ghrelin, a 28 residues acylated peptide, is the natural ligand of the growth-hormone secretagogue receptor (GHS-R), which also interacts with small synthetic peptides. We investigated the importance of each of the first 14 N-terminal residues by Ala replacement (Ala-scan) and also of the N-terminal positive charge, on the recombinant GHS-R expressed in HEK293 or CHO cells by binding, IP and Ca 2+ assays. Nearly all of the replacements had no significant effect on the ligand binding or IP 3/Ca 2+ stimulation. Exceptions were the modification of the N-terminal residue to [A 1]- or N α-acetyl-ghrelin (1–14), confirming the requirement for the positive charge at the amino-terminus. Mutation of [F 4]- to [A 4]- or [Y 4]-ghrelin (1–14), were detrimental suggesting direct interaction with the GHS-R. [A 8] and [Y 8] were more potent than ghrelin (1–14), implying that the naturally occurring Glu 8 residue may not be the optimal.