title: Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi.
abstract: The antiserum AS7 can specifically immunoprecipitate alpha-Gi from membrane extracts as well as from a mixture of purified alpha-Gi and alpha-Go as ascertained using [32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate alpha-Gi from hepatocytes labelled with 32P it was evident that alpha-Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of alpha-Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH-glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+-dependent, [32P]ADP-ribosylation of alpha-Gi in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of alpha-Gi in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves alpha-Gi in its holomeric state.
authors.affiliation: Institute of Biochemistry, University of Glasgow, Scotland.
authors.forename: N J
authors.initials: NJ
authors.lastname: Pyne
authors.forename: G J
authors.initials: GJ
authors.lastname: Murphy
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: M D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005934:Glucagon / Q000031:analogs & derivatives / Q000494:pharmacology*; D007106:Immune Sera; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D000936:Antigen-Antibody Complex; D007106:Immune Sera; C033953:glucagon, N(alpha)-trinitrophenyl-His(1)-homo-Arg(12)-; D005934:Glucagon; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate