Conformational and molecular modeling studies of the cholecystokinin-1 and -2 guanine nucleotide-binding regulatory protein-coupled receptors

Guanine nucleotide-binding regulatory protein-coupled receptors (GPCRs) comprise an important class of integral membrane proteins involved in the transduction of extracellular stimuli to the cell cytoplasm. Despite the ubiquitous nature of these receptors and their importance in intracellular signaling pathways, rhodopsin is the only GPCR whose structure is known in considerable detail. The intractable high resolution structure of GPCRs has lead to the development of alternative methods in which receptor fragments corresponding to complete or partial domains of the intact receptor are structurally characterized by NMR or X-ray crystallography.; To address the structural basis for ligand-specificity in the CCK ₁-R and CCK₂-R, the conformational features of the putative third extracellular loop and several residues from the adjoining sixth and seventh transmembrane domains of the CCK₁-R, CCK₁-R(329–357), and the CCK₂-R, CCK₂-R(352–379), were examined by high resolution NMR spectroscopy in the presence of DPC micelles. The receptor domains included 1–3 turns of the putative TM domains, to facilitate tethering of the receptor domains to the micelle surface. Both site-directed mutagenesis and photo-affinity cross-linking have identified intermolecular contacts in the EC3 domain of the CCK₁-R; however, intermolecular contacts to the EC3 domain of the CCK₂-R have not been established. On the basis of the intermolecular interactions implied in the CCK/CCK ₁-R complex and the high level of sequence identity in the EC3 of the CCK receptor subtypes (∼54%), the CCK₁-R(329–357) and CCK₂-R(352–379) receptor domains were selected for further scrutiny.; Addition of peptidic and nonpeptidic agonists to the EC3 receptor domains produced a number of intermolecular nuclear Overhauser enhancements and concentration dependent amide 1H chemical shift perturbations. The conformational preferences of the receptor domains and ligands were used, along with the intermolecular NOEs to delineate noncovalent interactions in molecular models of the ligand-receptor complexes. The molecular models were compared and contrasted to rationalize the observed differences in the ligand-binding modes. The binding mechanisms, topological orientations and intermolecular interactions in the ligand-receptor complexes were rationalized in terms of the available NMR, mutagenesis, photo-affinity cross-linking and structure activity relationship (SAR) data. Differences in the localization of the NMR-derived ligand-receptor domain contacts have been used in conjunction with molecular modeling to provide support for subtype-distinct ligand binding sites in the CCK₁ and CCK₂ receptors.