Molecular Basis of Agonist Binding to the Type A Cholecystokinin Receptor

Abstract

Abstract:The receptors for cholecystokinin (CCK) peptides are guanine nucleotide-binding protein-coupled receptors in the rhodopsin/b-adrenergic receptor family. The molecular basis of natural ligand binding to the type A CCK receptor has been studied using ligand structure-activity series, receptor mutagenesis, and photoaffinity labeling studies. These have focused attention on the extracellular loop and tail domains, with the most direct insights coming from intrinsic photoaffinity labeling studies. A model of the binding of CCK to this receptor is consistent with all these studies. This model places the carboxyl terminus of CCK adjacent to the amino-terminal tail outside of transmembrane segment 1, the midregion of the peptide adjacent to the third extracellular loop outside of transmembrane segment 7, and includes a chargecharge interaction between peptide residue tyrosine-sulfate 27 and the arginine residue in the second extracellular loop of the receptor in position 197.Understanding of the molecular basis of agonist binding to receptors and their activation is of great interest and importance for the rational design of receptor-active drugs. The type A cholecystokinin (CCK) receptor is a valid drug target, with particular relevance for agonist drugs that might induce satiety and that could have a role in the pharmacotherapy of obesity. Indeed, orally active small molecule drug candidates with agonist activity at this receptor are being developed (Aquino et al. 1996). A more detailed understanding of the conformation of the active complex of CCK receptor occupied by the natural full agonist ligand, CCK, would help to refine such candidates and to develop new candidate drugs.The CCK receptor is a member of the Class I rhodopsin/ b-adrenergic family of guanine nucleotide-binding protein (G protein)-coupled receptors. This molecule was first identified by affinity labeling using a CCK-like probe (Pearson & Miller 1987) and was later purified, partially sequenced, and had its cDNA cloned (Wank et al. 1992). This assignment to this receptor family is based on sequence homology primarily within the transmembrane domains of member receptors. Being a member of the Class I family provides a particular advantage for the CCK receptor, based on the recent successful solution of a high resolution crystal structure of rhodopsin (Palczewski et al. 2000). 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. Ligand structure-activity seriesThree types of studies can be particularly useful for understanding the conformation of the CCK receptor and...

Structural basis of cholecystokinin receptor binding and regulation

“…For this to be most informative, the photolabile ligand should be structurally related to the natural hormone whose binding is being investigated. For CCK, high affinity, biologically active photolabile analogues have been developed with sites of covalent attachment in six of seven positions within the CCK pharmacophore (Miller & Lybrand, 2002). To date, four of these have been used to establish predominant sites of labeling the CCK 1 receptor.…”

“…This includes structure-activity relationship studies of ligands and receptors, modifying the ligands during synthesis and typically modifying the receptors by mutagenesis (Miller & Lybrand, 2002). Types of mutagenesis include segmental deletions and site-specific modifications.…”

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

“…The binding site for this hormone at the CCK1R, its physiological target for induction of satiety, 8 is at the external surface of the lipid bilayer, with contributions by the amino-terminal tail and extracellular loop regions of this receptor. 34 This has been established by receptor mutagenesis and chimeric receptor analysis, 34,35 as well as by the direct approach of intrinsic photoaffinity labeling of receptor residues in spatial approximation with residues within the natural peptide ligand. 36 With the pharmacophoric region of CCK that is responsible for activity at the CCK1R representing the carboxylterminal heptapeptide amide, photoaffinity labeling of this receptor has been successful with probes incorporating a photolabile site of crosslinking at six of these seven positions within this portion of the peptide, as well as just beyond this region at the peptide amino terminus.…”

Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor

“…Whereas naturally occurring CCK peptides eight or more residues in length bind with high affinity and are potent activators of both the CCK1R and the CCK2R, gastrin and shorter CCK peptides are high affinity ligands and potent activators of the CCK2R only (18). The basis for CCK binding to CCK1R is particularly well defined, based on direct photoaffinity labeling spatial approximation constraints at seven positions within CCK-8, which show determinants distributed throughout the extracellular loop and amino-terminal tail regions but not within the predicted transmembrane domain bundle (12,19). In contrast, at the CCK2R, the carboxyl-terminal end of the peptide may be sited closer to the helical bundle (12,20,21).…”