PHOTOAFFINITY-LABELING OF RAT PANCREATIC CHOLECYSTOKININ TYPE-A RECEPTOR ANTAGONIST BINDING-SITES DEMONSTRATES THE PRESENCE OF A TRUNCATED CHOLECYSTOKININ TYPE-A RECEPTOR

During the past few years, several antagonist ligands for cholecystoki nin (CCK) receptors have been discovered, but the mechanism of action of these candidate drugs, as well as the nature of their molecular tar gets, remains poorly documented. In a previous study, we developed a n ew antagonist radioligand, I-125-Bolton-Hunter-labeled JMV-179, for th e CCK-A receptor (CCK-AR), to analyze CCK antagonist binding sites in pancreatic plasma membranes. We found that I-125-Bolton-Hunter-labeled JMV-179 identified 4 times as many sites as did an agonist radioligan d, although agonists were able to interact competitively with the enti re population of antagonist sites. In the present work, using biochemi cal approaches we have identified and characterized CCK antagonist bin ding sites in pancreatic plasma membranes. We synthesized the photoact ivable antagonist probe I-125-azidosalicylic acid (ASA)-JMV-179. The b inding of I-125- ASA-JMV-179 to plasma membranes was inhibited by JMV- 179 (IC50, 6 +/- 2 nM), by (Thr(28),Ahx(31))-CCK-25-33 (IC50, 1.2 +/- 0.5 nM), and by the nonpeptide CCK-AR antagonist L-364,718 (IC50, 2 a 1 nM). Photoaffinity labeling using pancreatic membranes or acini demo nstrated that I-125-ASA-JMV-179 detected a new 47-50-kDa protein in ad dition to the 85-100-kDa CCK-AR. The 47-50-kDa protein was not directl y detected by a photoactivable agonist, but agonists could inhibit its covalent labeling by I-125-ASA-JMV-179 (IC50 for (Thr(28),Ahx(31))-CC K-25-33, 15 nM). In competition assays using nonsolubilized or solubil ized membranes, this protein displayed binding features of the CCK-AR and was retained on immobilized wheat germ agglutinin, as was the CCK- AR. To further characterize the 47-50-kDa protein, deglycosylation and protease digestions were performed, and the digestion products were s eparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protease digestions of both the CCK-AR and the 47-50-kDa protein yiel ded identical labeled fragments, demonstrating a structural relationsh ip between the two proteins. The CCK-AR, which has three potential sit es for N-glycosylation on the amino-terminal extracellular domain and one on the second extracytoplasmic loop, was deglycosylated to a 42-kD a peptide. The 47-50-kDa protein was deglycosylated to a 35-kDa peptid e. These data, and the localization of the labeled fragments in the am ino acid sequence of the receptor, suggest that the 47-50-kDa protein represents a CCK-AR lacking its amino-terminal extracellular domain. T his study, with the first photoreactive antagonist probe for the CCK-A R, demonstrates that some of the heterogeneity of CCK antagonist bindi ng sites in pancreatic plasma membranes is due to the presence of a tr uncated CCK-AR.