Phosphorylation of cholecystokinin receptors expressed on Chinese hamster ovary cells. Similarities and differences relative to native pancreatic acinar cell receptors.
Phosphorylation of G protein-coupled receptors is an established mechanism for desensitization in response to agonist stimulation. We previously reported phosphorylation of the pancreatic acinar cell cholecystokinin (CCK) receptor and the establishment of two-dimensional phosphopeptide mapping of its sites of phosphorylation (Ozcelebi, F., and Miller, L. J. (1995) J. Biol. Chem. 270, 3435-3441). Here, we use similar techniques to map sites of phosphorylation of the same receptor expressed on a stable receptor-bearing Chinese hamster ovary (CHO)-CCKR cell line. Like the native cell, the CHO-CCKR cell receptor was phosphorylated in response to agonist stimulation in a concentration-dependent manner; however, the time course was quite different. CHO-CCKR cell receptor phosphorylation increased progressively to a plateau after 15 min, while in the acinar cell it peaks within 2 min and returns to baseline over this interval. There were distinct qualitative and quantitative differences in the sites of phosphorylation of the two receptor systems. One site previously attributed to action of a staurosporine-insensitive kinase in the acinar cell was absent in the CHO-CCKR cell. Site-directed mutagenesis was utilized to eliminate predicted sites of protein kinase C action, but only two of four such sites affected the phosphopeptide map of this receptor. Chemical and radiochemical sequencing were performed on these and other phosphopeptides which were present in both the CHO-CCKR cells and agonist-stimulated pancreatic acinar cells to provide direct evidence for the phosphorylation sites actually utilized. Thus, these data support the usefulness and limitations of a model cell system in studying receptor phosphorylation and desensitization.
Ozcelebi
F
F
Center for Basic Research in Digestive Diseases and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Rao
R V
RV
Holicky
E
E
Madden
B J
BJ
McCormick
D J
DJ
Miller
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002766:Cholecystokinin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D008961:Models, Structural; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012844:Sincalide / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D010748:Phosphopeptides; D011949:Receptors, Cholecystokinin; D011994:Recombinant Proteins; D002766:Cholecystokinin; D012844:Sincalide; D013755:Tetradecanoylphorbol Acetate