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.

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K i ‫؍‬ 8.9 ؎ 1.1 nM). It covalently labeled the CCK receptor either within the amino terminus (between Asn 10 and Lys 37 ) or within the third extracellular loop (Glu 345 ), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa 29 ) but resulted in elimination of the covalent attachment of the Bpa 24 probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.Guanine nucleotide-binding protein (G protein) 1 -coupled receptors represent a remarkable group of structurally homologous membrane proteins that can bind and be activated by widely diverse ligands. The molecular details of how ligands as structurally dissimilar as photons, biogenic amines, peptides, and glycoproteins can elicit similar conformational changes in the cytosolic face of their receptors (where G protein-coupling occurs) are far from clear. Our best understanding of this process relates to the smallest ligands that appear to bind within the confluence of helices within the lipid bilayer (1), where we have analogous low resolution crystal structures on which to rely (2, 3). As the ligands get larger and more structurally complex, the binding domains tend to move toward the extracellular face of the membrane, with extracellular loop and amino-terminal tail domains becoming more important (1, 4). These are receptor domains for which we have minimal meaningful structural data.We have been quite interested in the molecular basis of ligand binding to the type A cholecystokinin (CCK) receptor (5-10). This receptor is a member of the class I family of G protein-coupled receptors, along with rhodopsin and the ␤-adrenergic receptor (11). CCK occurs as a series of linear peptides, having lengths ranging from 8 to 58 residues (12). These all share their carboxyl-terminal domain, with the carboxylterminal h...

Background:Photoreceptors undergo degeneration when phototransduction is impaired. Results: The endoplasmic reticulum stress markers and processing of the associated caspases are elevated in retinas with cone photoreceptor CNG channel deficiency. Conclusion:The endoplasmic reticulum stress-associated apoptotic pathways play a crucial role in cone degeneration. Significance: Understanding of the mechanism(s) of photoreceptor degeneration is essential for development of therapeutic strategies.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca 2ϩ influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3