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 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.
Van Craenenbroeck
Mélanie
M
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.
Gregoire
Françoise
F
De Neef
Philippe
P
Robberecht
Patrick
P
Perret
Jason
J
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D054439:Ghrelin; D006801:Humans; D036361:Peptide Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins / Q000378:metabolism
D054439:Ghrelin; D036361:Peptide Hormones; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins; D000409:Alanine