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

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 Ca2+ assays. Nearly all of the replacements had no significant effect on the ligand binding or IP3/Ca2+ stimulation. Exceptions were the modification of the N-terminal residue to [A(1)]- or N-alpha-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. (C) 2004 Elsevier Inc. All rights reserved.