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

Abstract

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.

Insights into the Cholecystokinin 2 Receptor Binding Site and Processes of Activation

“…Several other reports implicated ECL2 in ligand specificity in aminergic and other small ligand receptors (Shi and Javitch, 2002). It is noteworthy that, in our molecular model, the ECL2 enters into the cavity formed by the seven helices, as observed for rhodopsin in the X-ray structure, to interact with the Cterminal part of CCK (Silvente-Poirot et al, 1999;Gales et al, 2003a). The fact that our molecular model and experimental data are in accordance with these data push us to pursue the characterization of the CCK binding site.…”

“…Based on these different results, we established a molecular model of the CCK2R occupied by CCK. This model enabled us to identify two new residues not identified by mutagenesis, Tyr189(Y4.60) and Asn358(N6.55), in interaction with the C-terminal Phe 9 -amide of CCK (Gales et al, 2003a). Mutagenesis of these residues and affinity studies with modified peptides allowed us to confirm these interactions and to show the involvement of the transmembrane domains (TM) 4 and 6, in addition to the ECL2 in the CCK binding site and receptor activation.…”

“…The WT-CCK2R and CCK-occupied WT-CCK2R molecular models were built as described previously (Gales et al, 2003a). In brief, to dock CCK into this CCK2R model, we took into account our previous mutagenesis data and imposed as constraint the direct contact point identified between the lateral side chains of His207 located in the second extracellular loop and Asp8 of CCK.…”

Partial Agonism, Neutral Antagonism, and Inverse Agonism at the Human Wild-Type and Constitutively Active Cholecystokinin-2 Receptors

“…For flexible docking of synthetic ligands, the CCK2R.CCK-4 complex was built using a CCK2R.CCK9 complex previously modeled and validated on the basis of site-directed mutagenesis data (Gales et al, 2003a;Langer et al, 2005). By doing so, a number of distinct positions with similar scoring functions were generated (data not shown).…”

“…The effect of mutation of four reference amino acids (Blaker et al, 1998;Gales et al, 2003a;Langer et al, 2005) of the CCK binding site on the affinity of synthetic ligands was evaluated for docking refinement. Mutation factors, which account for shifts of affinity, varied for the different nonpeptide ligands and were generally lower than for CCK-4 ( Table 1).…”