Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.
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 I3B 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 microM) 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.
Bushfield
M
M
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Murphy
G J
GJ
Lavan
B E
BE
Parker
P J
PJ
Hruby
V J
VJ
Milligan
G
G
Houslay
M D
MD
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000008:administration & dosage / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000804:Angiotensin II / Q000008:administration & dosage / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005934:Glucagon / Q000008:administration & dosage / Q000494:pharmacology; D006728:Hormones / Q000008:administration & dosage / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000032:analysis; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000008:administration & dosage / Q000494:pharmacology
D000596:Amino Acids; D006728:Hormones; D009943:Organophosphorus Compounds; D014667:Vasopressins; D000804:Angiotensin II; D015124:8-Bromo Cyclic Adenosine Monophosphate; D005934:Glucagon; D011493:Protein Kinase C; D019204:GTP-Binding Proteins