Refinement of the Conformation of a Critical Region of Charge-Charge Interaction between Cholecystokinin and Its Receptor

Abstract

Insight into the molecular basis of cholecystokinin (CCK) binding to its receptor has come from receptor mutagenesis and photoaffinity labeling studies, with both contributing to the current hypothesis that the acidic Tyr-sulfate-27 residue within the peptide is situated adjacent to basic Arg 197 in the second loop of the receptor. Here, we refine our understanding of this region of interaction by examining a structure-activity series of these positions within both ligand and receptor and by performing three-dimensional molecular modeling of key pairs of modified ligand and receptor constructs. The important roles of Arg 197 and Tyr-sulfate-27 were supported by the marked negative impact on binding and biological response with their natural partner molecule when the receptor residue was replaced by acidic Asp or Glu and when the peptide residue was replaced by basic Arg, Lys, p-amino-Phe, p-guanidino-Phe, or p-methylamino-Phe. Complementary ligand-receptor chargeexchange experiments were unable to regain the lost function.This was supported by the molecular modeling, which demonstrated that the charge-reversed double mutants could not form a good interaction without extensive rearrangement of receptor conformation. The models further predicted that R197D and R197E mutations would lead to conformational changes in the extracellular domain, and this was experimentally supported by data showing that these mutations decreased peptide agonist and antagonist binding and increased nonpeptidyl antagonist binding. These receptor constructs also had increased susceptibility to trypsin degradation relative to the wild-type receptor. In contrast, the relatively conservative R197K mutation had modest negative impact on peptide agonist binding, again consistent with the modeling demonstration of loss of a series of stabilizing inter-and intramolecular bonds. The strong correlation between predicted and experimental results support the reported refinement in the three-dimensional structure of the CCK-occupied receptor.

Cholecystokinin and Gastrin Receptors

“…3) (193,228). Accordingly, a model of binding of CCK to the CCK1R was proposed in which the COOH terminus of CCK and the tyrosine sulfate were in interaction with Trp39 and Arg197, respectively, and the NH 2 -terminal moiety was in contact with the third extracellular loop of the receptor (132). This second model of CCK positioning into the CCK1R binding site is divergent from that obtained on the basis of site-directed mutagenesis results (16,237).…”

The Crystallographic Model of Rhodopsin and Its Use in Studies of Other G Protein–Coupled Receptors

“…However, it is often difficult to assess the quality and relevance of molecular models. Concerns about the structures of the loops in rhodopsin have been expressed (21,69), as have questions about the validity of the rhodopsin structure as a general template for GPCRs (25,76,77).…”

Molecular Basis of Agonist Binding to the Type A Cholecystokinin Receptor

“…Attempts to apply this experimental approach to the CCK receptor have been unable to reestablish high affinity binding or potent biological activity (Gigoux et al 1999). This is true even when dealing with a site that is well established as a position of chargecharge interaction between the acidic tyrosine-sulfate in po-sition 27 of the CCK peptide and basic Arg 197 within the second extracellular loop region of this receptor (Ding et al 2002).…”

“…It is quite likely that there will be substantial similarity between the conformation of transmembrane helices and bundle formation within the membrane of the CCK receptor and rhodopsin. However, in a recent report, it was demonstrated that the loop and tail positions in rhodopsin are not rel- evant to analogous portions of the CCK receptor (Ding et al 2002). Homology modeling of the CCK receptor for these regions of rhodopsin yield no binding cleft of adequate size and reasonable position to accommodate the natural peptide ligand.…”

“…evant to analogous portions of the CCK receptor (Ding et al 2002). Homology modeling of the CCK receptor for these regions of rhodopsin yield no binding cleft of adequate size and reasonable position to accommodate the natural peptide ligand.…”