title: Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.
abstract: Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
authors.forename: E S
authors.initials: ES
authors.lastname: Tobias
authors.forename: E
authors.initials: E
authors.lastname: Rozengurt
authors.forename: J M
authors.initials: JM
authors.lastname: Connell
authors.forename: M D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D000818:Animals; D019556:COS Cells; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054770:G-Protein-Coupled Receptor Kinase 3; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D008297:Male; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D018027:Receptors, Glucagon / Q000096:biosynthesis* / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D018027:Receptors, Glucagon; D005934:Glucagon; D000242:Cyclic AMP; C089212:protein kinase D; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054770:G-Protein-Coupled Receptor Kinase 3; C517851:Grk3 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D013755:Tetradecanoylphorbol Acetate