Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminaI probe

Affinity labeling is a powerful tool to establish spatial approximations be tween photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenyla lanine (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 amin o terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosyl ation, micropurification, and Edman degradation sequencing. Truncation of t he receptor to eliminate residues 1-30 had no detrimental effect on CCK bin ding, stimulated signaling, or affinity labeling through a residue within t he pharmacophore (Bpa(29)) but resulted in elimination of the covalent atta chment of the Bpa(24) probe to the receptor, Thus, the distal amino terminu s of the CCK: receptor resides above the docked ligand, compressing the por tion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellu lar 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 coun terclockwise organization of the transmembrane helical domains.