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

S A Rosenzweig,J D Jamieson,L J Miller

doi:10.1083/jcb.96.5.1288

Abstract

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 125-I-CCK-33 for 15 min at 23 degrees C followed by washing and cross-linking with disuccinimidyl suberate. Specific labeling of a major Mr 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of Mr greater than 200,000, Mr 130,000-140,000, and, Mr 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 (Kd approximately 9 nM). The Mr 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 microM) for 2 or 15 min at 37 degrees C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that approximately 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 Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.

Highlights

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
CCK-33 contains three primary amino groups located at positions Lysl or Lys1~ [2]
The results indicate that the concentration of CCK-8 causing half-maximal inhibition of affinity labeling of the Mr 85,000 protein was 9 nM (Fig. 6, lower panel)

Summary

Introduction

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. 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 Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell. As with other polypeptide hormones, it is generally accepted that the initial step in CCK action is its binding to specific plasmalemmal receptors [7] This is supported by recent binding studies

Methods

Results

Conclusion