Refinement of the Structure of the Ligand-occupied Cholecystokinin Receptor Using a Photolabile Amino-terminal Probe

Abstract

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...

The Biologically Crucial C Terminus of Cholecystokinin and the Non-peptide Agonist SR-146,131 Share a Common Binding Site in the Human CCK1 Receptor

“…Mutation of candidate residues and extensive characterization of the resulting mutants allowed us to position the C-terminal biological part of CCK in hydrophobic pockets formed by aromatic and nonaromatic amino acids located in the upper half of transmembrane helices III, V, VI, and VII. Our data, therefore, refute the model of the CCK1R⅐CCK complex proposed by other investigators in which the C terminus of CCK interacts with an amino acid residue (Trp-39) of helix I (18). Furthermore, binding site for the non-peptide agonist SR-146,131 was identified and experimentally validated as overlapping with that of the C-terminal tripeptide of CCK.…”

“…This docking mode of the C terminus of CCK into CCK1R is further supported by an NMR study of the interactions between CCK and a fragment of CCK1R comprising the top portion of helix VI and the third extracellular loop (31). On the other hand, it differs from that derived from photoaffinity labeling studies (18,32,33). In the photoaffinity labeling experiments with a CCK photoprobe in which the C-terminal Phe residue was replaced by benzophenylalanine, Trp-39 at the top of helix I was identified, supporting the hypothesis that the C terminus of CCK was in close proximity of the first helix (32).…”

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

“…Mapping-We have successfully used CNBr for identification of ligand binding sites of the cholecystokinin receptor (26,32,33), the secretin receptor (11, 12, 14 -17), and the motilin receptor (34). Here again, we used CNBr as the first indication of domain of labeling for the calcitonin receptor.…”

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

“…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. 37 All of these points of experimentally established spatial approximation with specific residues within the CCK1R have been accommodated into working models of the CCK-occupied CCK1R. 36 This has even been further constrained with a series of fluorescent analogs of CCK and use of fluorescence resonance energy transfer with a series of fluorescently tagged CCK1R constructs to elucidate and apply 12 distance constraints to this model.…”