Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: A biochemical and autoradiographic study

Using the combined approaches of affinity labeling and light and electron microscopic autoradiography, we investigated the identification and localization of cholecystokinin (CCK)-binding sites on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, we incubated rat pancreatic plasma membranes with ¹²⁵I-CCK-33 for 15 min at 23°C followed by washing and cross-linking with disuccinimidyl suberate. Specific labeling of a major M(r) 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of M(r) > 200,000, M(r) 130,000-140,000, and M(r) 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (K(d) ~9 nM). The M(r) 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with ¹²⁵I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 μM) for 2 or 15 min at 37°C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that ~60% of the total grains were located within ±1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. From these data, it was estimated that each acinar cell possesses at least 5,000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. Together, these data indicate that CCK interacts with a M(r) 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.