Hormonal regulation of Gi2 α-subunit phosphorylation in intact hepatocytes

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Abstract
Hepatocytes contain the Gi2 and Gi3 forms of the ''Gi-family'' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and 13B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-.alpha.-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 .alpha.-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5''-[.beta..gamma.-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity (''Gi''-function). The immunoprecipitation of phosphorylated Gi-2.alpha.-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 Gz (a''Gi-like'' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the .alpha.-subunits of either Gi3 or Gs was observed. .alpha.-Gi2 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 .alpha.-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of .alpha.-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of .alpha.-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 .mu.M) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated .alpha.-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [.gamma.-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated .alpha.-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of .alpha.-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of .alpha.-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

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