Hepatocytes contain the G(i)2 and G(i)3 forms of the 'G(i)-family' of guanine-nucleotide-binding proteins (G-proteins), but not G(i)1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate G(i)2 and G(i)3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein G(s). Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-α-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the α-subunit of immunoprecipitated G(i)2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[βγ-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('G(i)'-function). The immunoprecipitation of phosphorylated G(i)-2α-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of G(z) (a 'G(i)-like' G protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the G(i) family), in the immunoprecipitation assay. No labelling of the α-subunits of either G(i)3 or G(s) was observed. α-G(i)2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of α-G(i)2 was markedly biphasic where the loss of G(i) function paralleled the high-affinity component of the labelling of α-G(i)2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of α-G(i)2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 μM) or 8-bromo-cyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated α-G(i)2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [γ-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated α-G(i)2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of α-G(i)2 in intact cells. We suggest that there are two possible sites for the phosphorylation of α-G(i)2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.
