Abstract

During the past few years, several antagonist ligands for cholecystokinin (CCK) receptors have been discovered, but the mechanism of action of these candidate drugs, as well as the nature of their molecular targets, remains poorly documented. In a previous study, we developed a new antagonist radioligand, 125I-Bolton-Hunter-labeled JMV-179, for the CCK-A receptor (CCK-AR), to analyze CCK antagonist binding sites in pancreatic plasma membranes. We found that 125I-Bolton-Hunter-labeled JMV-179 identified 4 times as many sites as did an agonist radioligand, although agonists were able to interact competitively with the entire population of antagonist sites. In the present work, using biochemical approaches we have identified and characterized CCK antagonist binding sites in pancreatic plasma membranes. We synthesized the photoactivable antagonist probe 125I-azidosalicyclic acid (ASA)-JMV-179. The binding of 125I-ASA-JMV-179 to plasma membranes was inhibited by JMV-179 (IC50, 6 +/- 2 nM), by (Thr28, Ahx31)-CCK-25-33 (IC50, 1.2 +/- 0.5 nM), and by the nonpeptide CCK-AR antagonist L-364,718 (IC50, 2 +/- 1 nM). Photoaffinity labeling using pancreatic membranes or acini demonstrated that 125I-ASA-JMV-179 detected a new 47-50-kDa protein in addition to the 85-100-kDa CCK-AR. The 47-50-kDa protein was not directly detected by a photoactivable agonist, but agonists could inhibit its covalent labeling by 125I-ASA-JMV-179 (IC50 for (Thr28,Ahx31)-CCK-25-33, 15 nM). In competition assays using nonsolubilized or solubilized 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 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protease digestions of both the CCK-AR and the 47-50-kDa protein yielded identical labeled fragments, demonstrating a structural relationship between the two proteins. The CCK-AR, which has three potential sites for N-glycosylation on the amino-terminal extracellular domain and one on the second extracytoplasmic loop, was deglycosylated to a 42-kDa peptide. The 47-50-kDa protein was deglycosylated to a 35-kDa peptide. These data, and the localization of the labeled fragments in the amino acid sequence of the receptor, suggest that the 47-50-kDa protein represents a CCK-AR lacking its amino-terminal extracellular domain.(ABSTRACT TRUNCATED AT 400 WORDS)