Binding of a phenethyl ester analogue of cholecystokinin to the solubilized pancreatic cholecystokinin receptor: use in ligand-affinity chromatography.
Pancreatic acinar cells have both high and low affinity receptors for cholecystokinin (CCK), yet their membranes appear to possess only a single class of binding sites. Recently, gallbladder membrane CCK receptors were shown to undergo inter-conversion between two affinity states dependent on G protein coupling. Keys for that observation were the differential binding affinities of CCK and a phenethyl ester analogue of CCK (OPE), with the high affinity state binding CCK with higher affinity than OPE, and the low affinity state binding OPE with higher affinity than CCK. Here, we performed analogous experiments using these ligands and both pancreatic membranes and a solubilized preparation. Both preparations were found to have only single affinity states of this receptor. However, the state on membranes had a higher affinity for CCK than for OPE, and that on the solubilized preparation had a higher affinity for OPE than for CCK. This supports the hypothesis that the ternary complex of ligand-receptor-G protein found in membranes represents the high affinity state of this receptor, while the uncoupled form of this receptor after solubilization represents its low affinity state. The high affinity of OPE for the solubilized receptor can be utilized in a purification strategy to follow receptor-bearing fractions and to provide an efficient and specific affinity-binding step.
Miller
L J
LJ
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, MN 55905.
Hadac
E M
EM
Gates
L K
LK
Gaisano
H Y
HY
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000031:analogs & derivatives*; D002846:Chromatography, Affinity / Q000379:methods*; D008297:Male; D010179:Pancreas / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification* / Q000378:metabolism; D012844:Sincalide / Q000031:analogs & derivatives* / Q000378:metabolism
D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; C067408:cholecystokinin (26-32), Tyr-Gly-Nle(28,31) phenethyl ester-; D002766:Cholecystokinin; D012844:Sincalide