Received for publication August 14, 2006.
Revised September 21, 2006.
Accepted for publication September 22, 2006.

INSIGHTS INTO THE CHOLECYSTOKININ 2 RECEPTOR BINDING SITE AND PROCESSES OF ACTIVATION

Abstract

The cholecystokinin 2 receptor (CCK2R) appears as a pharmacological target for the treatment of many major diseases. To complete the mapping of the CCK2R binding site and its activation processes, we have looked for the receptor residues that interact with Trp⁶, an essential residue for CCK binding and activity. In the molecular model of the CCK-occupied CCK2R that we built, the indole group of Trp⁶ stacked with the phenyl ring of Phe120 (ECL1) and interacted with the imidazole group of His381(H7.39) and the phenyl ring of Tyr385(H7.43). Mutagenesis and pharmacological studies validated these interactions. Importantly, the mutation of Phe120 to Trp conferred constitutive activity to the CCK2R. Molecular modeling and experimental works identified the residues involved in the activation cascade initiated by Trp⁶ and revealed that the constitutively active F120W mutation mimics the conformational changes induced by Trp⁶ resulting in : i) the exposure of Glu151(E3.49) of the conserved E/DRY motif ii) the formation of an amphiphatic pocket involving protonated Glu151(E3.49) and Leu330 (ICL3) iii) the opening of the intracellular loops 2 and 3 and the release of Arg158 (ICL2). The R158A mutation was shown to affect inositol phosphate production while the E151A and L330E mutations induced constitutive inositol phosphate production. Given that a constitutively active variant of the CCK2R has been identified in different cancers and the fact that the E151A mutant has been reported to induce tumours, these studies should help in the development of potent inverse agonists to inhibit the constitutive activation of the CCK2R.

Key words: CCK/gastrin, Gq/11 family, Phospholipase C's, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches, Receptor binding studies, Peptide hormones

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