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Abstract:
The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein- coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and biological activity, and to determine whether CCK and the selective non-peptide agonist, SR-146,131, share a common binding site. Identification of putative amino acids of the CCK1R binding site was achieved by dynamics-based docking of the ligand CCK in a refined three-dimensional model of the CCK1R using, as constraints, previous results that identified contact points between residues of CCK and CCK1R (Kennedy, K., Gigoux, V., Escrieut, C., Maigret, B., Martinez, J., Moroder, L., Frehel, D., Gully, D., Vaysse, N., and Fourmy, D. (1997) J. Biol. Chem 272, 2920-2926 and Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem 274, 20457-20464). By this approach, a series of residues forming connected hydrophobic clusters were identified. Pharmacological and functional analysis of mutated receptors indicated that a network of hydrophobic residues including Cys- 94, Met-121, Val-125, Phe-218, Ile-329, Phe-330, Trp-326, Ile- 352, Leu-356, and Tyr-360, is involved in the binding site for CCK and in the activation process of the CCK1R. Within this hydrophobic network, the physico-chemical nature of residue 121 seems to be essential for CCK1R functioning. Finally, the biological properties of mutants together with dynamic docking of SR-146,131 in the CCK1R binding site demonstrated that SR- 146,131 occupies a region of CCK1R binding site which interacts with the C-terminal amidated tripeptide of CCK, i.e. Met-Asp- Phe-NH2. These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands.
