Biochemical Characterization of a Subtype Pancreatic Cholecystokinin Receptor and of its Agonist Binding Domain

Abstract

This study was undertaken in order to improve photoaffinity labelling efficiency of pancreatic cholecystokinin receptor by the cleavable probe ¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33 and to further characterize the denaturated receptor and is agonist binding domain. Membrane bound pancreatic cholecystokinin receptor was specifically labelled by ¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33 as a component of Mr ≈ 85,000-100,000. The efficiency of the photolabelling was 3–4%. Performing photolysis on [¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33-receptor] complexes solubilized by CHAPS did not affect specificity of the labelling reaction but enhanced its efficiency so that up to 10% of the receptor site population could be cross-linked. Several lectins were tested for their ability to recognize and purify the cholecystokinin receptor denaturated by Nonidet P-40. Wheat germ agglutinin provided the best recovery and purification rate. The receptor was fully adsorbed on immobilized wheat germ agglutinin, while only a fraction was retained on ricin II (28%) and Ulex europaeus (28%), thus suggesting that the receptor is heterogeneously glycosylated. Finally, major labelled receptor fragments were generated by enzymatic digestion. There were: endoproeinase Glu-Mr → C ≈ 34,000; endoproteinase Glu-C/trypsin → Mr ≈ 12,000; chymotrypsinlendoproteinase Glu-C → Mr ≈ 16,000 and 12,000. The fragment of Mr 2 34,000 was deglycosylated to a component of Mr ≈ 22,000 whereas the other fragments were insensitive to deglycosylation Such results strongly suggest that cholecystokinin binding occurs in a non-glycosylated domain of the cholecystokinin receptor protein.