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

• Published in: The Journal of biological chemistry Vol. 276; no. 6; pp. 4236 - 4244
• Main Authors: Ding, Xi-Qin, Dolu, Vesile, Hadac, Elizabeth M., Holicky, Eileen L., Pinon, Delia I., Lybrand, Terry P., Miller, Laurence J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.02.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; CHO Cells; Cricetinae; Hydrolysis; Ligands; Models, Molecular; Molecular Sequence Data; Photoaffinity Labels; Photochemistry; Protein Conformation; Receptors, Cholecystokinin - chemistry; Receptors, Cholecystokinin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003798200

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 (Ki = 8.9 ± 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn10 and Lys37) or within the third extracellular loop (Glu345), 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 (Bpa29) but resulted in elimination of the covalent attachment of the Bpa24 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.