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.
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.
Pyne
N J
NJ
Institute of Biochemistry, University of Glasgow, Scotland.
Murphy
G J
GJ
Milligan
G
G
Houslay
M D
MD
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*
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