Vol. 63, Issue 5, 973-982, May 2003

Identification of Tyrosine 189 and Asparagine 358 of the Cholecystokinin 2 Receptor in Direct Interaction with the Crucial C-Terminal Amide of Cholecystokinin by Molecular Modeling, Site-Directed Mutagenesis, and Structure/Affinity Studies

Céline Galés, Marc Poirot, Julien Taillefer, Bernard Maigret, Jean Martinez, Luis Moroder, Chantal Escrieut, Lucien Pradayrol, Daniel Fourmy, and Sandrine Silvente-Poirot

Institut National de la Santé et de la Recherche Médicale (INSERM) U 531, Institut Louis Bugnard, CHU Rangueil, Toulouse, France (C.G., J.T., C.E., L.P., D.F.); INSERM U 563, Département Innovation Thérapeutique et Oncologie Moléculaire, Institut Claudius Regaud, Toulouse, France (M.P.); Laboratoire de Chimie Théorique, Université de Nancy, Vandoeuvre, Nancy, France (B.M.); Centre National de la Recherche Scientifique-Unité de Recherche Associée 1845, Faculté de Pharmacie, Montpellier, France (J.M.); and Max Planck Institute für Biochemie, Martinsried, Germany (L.M.).

The cholecystokinin (CCK) receptors CCK1R and CCK2R exert important central and peripheral functions by binding the neuropeptide cholecystokinin. Because these receptors are potential therapeutic targets, great interest has been devoted to the identification of efficient ligands that selectively activate or inhibit these receptors. A complete mapping of the CCK binding site in these receptors would help to design new CCK ligands and to optimize their properties. In this view, a molecular model of the CCK2R occupied by CCK was built to identify CCK2R residues that interact with CCK functional groups. No such study has yet been reported for the CCK2R. Docking of CCK in the receptor was performed by taking into account our previous mutagenesis data and by using, as constraint, the direct interaction that we demonstrated between His207 in the CCK2R and Asp8 of CCK (Mol Pharmacol 54:364-371, 1998; J Biol Chem 274:23191-23197, 1999). Two residues that had not been revealed in our previous mutagenesis studies, Tyr189 (Y4.60) and Asn358 (N6.55), were identified in interaction via hydrogen bonds with the C-terminal amide of CCK, a crucial functional group of the peptide. Mutagenesis of Tyr189 (Y4.60) and Asn358 (N6.55) as well as structure-affinity studies with modified CCK analogs validated these interactions and the involvement of both residues in the CCK binding site. These results indicate that the present molecular model is an important tool to identify direct contact points between CCK and the CCK2R and to rapidly progress in mapping of the CCK2R binding site. Moreover, comparison of the present CCK2R.CCK molecular model with that of CCK1R.CCK, which we have previously published and validated, clearly argues that the positioning of CCK in these receptors is different.