Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells.
The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
Ozcelebi
F
F
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Miller
L J
LJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000494:pharmacology*; D003488:Cyanogen Bromide; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009281:Naphthalenes; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D009281:Naphthalenes; D010748:Phosphopeptides; D011083:Polycyclic Compounds; D011949:Receptors, Cholecystokinin; D000255:Adenosine Triphosphate; D002217:Carbachol; D002766:Cholecystokinin; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D003488:Cyanogen Bromide