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

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.

Multiple extracellular loop domains contribute critical determinants for agonist binding and activation of the secretin receptor.

Distinct themes exist for ligand-binding domains of G protein-coupled receptors. The secretin receptor is prototypic of a recently described family in this superfamily which binds moderate-sized peptides possessing a diffuse pharmacophore. We recently demonstrated the importance of the N terminus and first loop of this receptor for secretin binding (Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) J. Biol. Chem. 270:14394-14398). Here, we extend those findings to define another receptor domain important for agonist recognition and to focus on critical determinants within each of these domains. Extending the secretin-vasoactive intestinal polypeptide (VIP) chimeric receptor approach, we confirmed and refined the critical importance of the N terminus and the need to complement this with other domains of the secretin receptor. There was redundancy in the complementary determinants required, with the second extracellular loop able to compensate for the absence of the first loop. The first 10 residues of the N terminus of the secretin receptor were critical. Sequential segmental and site replacements permitted focusing on the His189-Lys190 sequence at the C terminus of the first extracellular loop, and on four residues (Phe257, Leu258, Asn260, and Thr261) in the N-terminal half of the second loop as providing critical determinants. All receptor constructs which expressed sensitive cAMP responses to secretin (EC50 <5 nM) bound this peptide with high affinity. Of note, one construct dissociated high affinity binding of secretin from its biological responsiveness, providing a clue to the conformational "switch" that activates this receptor.