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 G i

Abstract

The antiserum AS7 can specifically immunoprecipitate α-G i from membrane extracts as well as from a mixture of purified α-G i and α-G o. as ascertained using [ 32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate α-G i from hepatocytes labelled with 32P it was evident that α-G i 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 α-G i. This was accompanied by the loss of inhibitory effect of G i 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 α-G i in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of α-G i in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of G i, it may be that phosphorylation leaves α-G i in its holomeric state.