Abstract

Understanding the structures of active and inactive agonist- and antagonist-bound receptor complexes is of great interest. In this work, we focus on position 30 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor. For this, we developed two photoaffinity labeling probes, replacing the naturally occurring tryptophan with p-benzoyl-l-phenylalanine (Bpa) or p-nitro-phenylalanine (NO₂-Phe). The Bpa probe was shown to represent an antagonist, whereas the NO₂-Phe probe stimulated intracellular calcium as a fully efficacious agonist (EC₅₀ = 81 ± 15 nM). Both ligands bound to the receptor specifically, although with lower affinity than CCK (K_i values: Bpa probe, 270 ± 72 nM; NO₂-Phe probe, 180 ± 40 nM). Both probes covalently labeled the receptor in an efficient manner. The Bpa antagonist labeled the receptor in two distinct regions as identified by cyanogen bromide cleavage, with labeled bands migrating at M_r = 25,000 and 4500. The former represented the glycosylated amino-terminal fragment, with the site of attachment further localized by endoproteinase Lys-C cleavage to the region between Asn¹⁰ and Lys³⁷. The latter was shown to represent the first extracellular loop using further cleavage and sequencing of the wild-type and a mutant receptor. Following the same approach, the NO₂-Phe agonist probe was shown to also label the first extracellular loop region. Radiochemical sequencing identified that the Bpa antagonist probe labeled receptor residue Lys¹⁰⁵, whereas the NO₂-Phe agonist probe labeled residue Leu⁹⁹. These data extend our understanding of the molecular basis of binding and the conformational states of this important receptor.