Biochemical characterization of a subtype pancreatic cholecystokinin receptor and of its agonist binding domain.
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 its agonist binding domain. Membrane bound pancreatic cholecystokinin receptor was specifically labelled by 125I-ASD-(Thr28,Ahx31)-CCK-25-33 as a component of Mr approximately 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 (58%), thus suggesting that the receptor is heterogeneously glycosylated. Finally, major labelled receptor fragments were generated by enzymatic digestion. There were: endoproteinase Glu-C----Mr approximately 34,000; endoproteinase Glu-C/trypsin----Mr approximately 12,000; chymotrypsin/endoproteinase Glu-C----Mr approximately 16,000 and 12,000. The fragment of Mr approximately 34,000 was deglycosylated to a component of Mr approximately 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.
Poirot
S S
SS
INSERM U 151, CHU Rangueil, Toulouse.
Dufresne
M
M
Jiménez
J
J
Vaysse
N
N
Fourmy
D
D
D000345:Affinity Labels; D000818:Animals; D002918:Chymotrypsin / Q000494:pharmacology; D006031:Glycosylation; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000032:analysis* / Q000187:drug effects / Q000302:isolation & purification
D000345:Affinity Labels; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; D002918:Chymotrypsin