The biochemical characterization of the native pancreatic cholecystokinin receptor using affinity labeling approaches.
Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using "intrinsic" probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a "topographical mapping" probe, which incorporates a flexible spacer of variable length between a CCK-like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other.(ABSTRACT TRUNCATED AT 250 WORDS)
Miller
L J
LJ
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905.
D000345:Affinity Labels; D000818:Animals; D002766:Cholecystokinin / Q000031:analogs & derivatives*; D007202:Indicators and Reagents / Q000378:metabolism; D010179:Pancreas / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification* / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism
D000345:Affinity Labels; D007202:Indicators and Reagents; D011949:Receptors, Cholecystokinin; D013388:Succinimides; C022581:Bolton-Hunter reagent; D002766:Cholecystokinin; D012844:Sincalide