CDB15:0000180 CCK — CCKAR

ABSTRACT: The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.

ABSTRACT: The interaction of the C-terminal octapeptide of cholecystokinin, CCK-8, with the third extracellular loop of human cholecystokinin-A receptor, CCK(A)-R(329-357), has been probed by high-resolution NMR and extensive computer simulations. The structure of CCK(A)-R(329-357) in the presence of dodecylphosphocholine micelles consists of three alpha-helices, with the first and third corresponding to the extracellular ends of transmembrane (TM) helices 6 and 7. The central helix, residues W335-R345, is found to lie on the zwitterionic surface. Titration with CCK-8 produces a stable complex with a number of intermolecular NOEs between the C-terminus of the ligand (Trp(30), Met(31), Asp(32)) and the interface of TM6 and the third extracellular loop (N333, A334, Y338) of the receptor fragment. The mode of ligand binding based on these intermolecular NOEs is in agreement with a number of published findings from receptor mutagenesis and photoaffinity cross-linking. Utilizing these ligand/receptor points of interaction, the structural features of CCK(A)-R(329-357), and also the structures of CCK-8 and CCK(A)-R(1-47) previously determined, extensive molecular dynamics simulations of the CCK-8/CCK(A)-R complex were carried out. The results provide unique insight into the molecular interactions and forces important for the binding of CCK-8 to CCK(A)-R.

ABSTRACT: A region between residues 38 and 42 of the human cholecystokinin-A (CCK-A) receptor was shown to be involved in the binding of CCK but not in that of JMV 179 and JMV 180, two peptides closely related to CCK (Kennedy, K., Escrieut, C., Dufresne, M., Clerc, P., Vaysse, N., and Fourmy, D. (1995) Biochem. Biophys. Res. Commun. 213, 845-852). In the present study, we have identified the residues of both the receptor and the ligand responsible for this differential binding. Residues Trp-39 and Gln-40 of the receptor were crucial for binding of the C-terminal nonapeptide of CCK as W39F and Q40N mutants demonstrated parallel decreases in both affinity and potency to induce accumulation of inositol phosphates (12.9- and 20.9-fold). The W39F and Q40N mutant receptors bound CCK analogues modified at their C-terminal end, including JMV 179 and JMV 180, as well as the C-terminal amidated heptapeptide of CCK, with identical affinities to the wild-type receptor. In contrast, W39F and Q40N mutants bound CCK octapeptide with the same decreased affinity as the CCK nonapeptide. The modeling of the CCK-A receptor and the docking of the peptide agonists [Thr,Nle]CCK9 and CCK-8 indicated that their N terminus was connected to the receptor through a strong bond network involving Trp-39 and Gln-40 thus confirming experimental data. These first molecular data identifying the agonist binding site of the human CCK-A receptor represent an important step toward the complete delineation of the agonist binding site and the understanding of the molecular mechanisms that govern differential activation of this receptor by CCK-related peptides.

ABSTRACT: Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2. 8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10- and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation.