Ala-scan of ghrelin (1-14): interaction with the recombinant human ghrelin receptor |
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Authors: | Van Craenenbroeck Mélanie Gregoire Françoise De Neef Philippe Robberecht Patrick Perret Jason |
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Institution: | Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Bat G/E, CP 611, 808 route de Lennik, B-1070 Brussels, Belgium. |
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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(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. |
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