Abstract
The present study was undertaken to determine if the cholecystokinin (CCK) receptor may be phosphorylated, and to gain insight into its regulation. For this, the ATP pool of rat pancreatic acini was prelabeled with 32P, and the cells were stimulated with various secretagogues. CCK receptors from treated cells were enriched by sequential fractionation to produce plasmalemma, and subsequent solubilization and lectin-affinity chromatography. This protocol detected a phosphorylated Mr = 85,000-95,000 plasma membrane glycoprotein with features similar to the CCK receptor. Phosphorylation of this protein occurred rapidly (less than 2 min) and in a concentration-dependent manner in response to CCK, and was inhibited by the CCK receptor antagonist L-364,718. Further evidence that this represented the CCK receptor included comigration of phosphorylated and CCK radioligand affinity-labeled proteins on sodium dodecyl sulfate-polyacrylamide gels, both in native forms and after endoglycosidase F deglycosylation, and the specific adsorption of the phosphoprotein to a CCK analogue affinity resin. Phosphorylation occurred predominantly on serine residues of the receptor protein. Phosphorylation of this protein was also enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in response to direct activation of this enzyme by 12-O-tetradecanoylphorbol 13-acetate. Thus, the pancreatic CCK receptor is phosphorylated in a regulated manner, in response to both homologous and heterologous secretagogues, and to protein kinase C activation.
Highlights
From the GastroenterologyBasic Research Unit, Mayo Clinicand Foundation, Rochester, Minnesota 55905 and the §West Haven Veterans Administration Hospital, West Haven, Connecticut06510
A protocol was developed for the rapid enrichment of this receptor while minimizing proteolysis and dephosphorylation
Phosphorylation occurredpredominantly on serine residues of the receptor protein. Phosphorylation of this protein was enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in MATERIALS AND METHODS
Summary
Peptides and Reagents-Synthetic CCK-8 (CCK-26-33) was purchased from Peninsula Laboratories (Belmont, CA). Plasma membrane receptors are uniquely situated toregu- creatic acini were washed and suspended in medium, containing 25 late cellular responsiveness to peptide hormones. Noylphorbol 13-acetate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethll TOwhom correspondence and reprint requests should be ad- anesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic dressed Mayo Clinic, Gastroenterology Basic Research Unit, Gug- acid; SDS, sodium dodecyl sulfate; UEA-I, Ulex europaeus aglutinin genheim 17, Rochester, MN 55905. A fraction enriched for plasma membranes was collected a t the interface between the sucrose layers and washed. This fraction was either directly separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography
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