Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes

M. Bushfield, G. J. Murphy, B. E. Lavan, P. J. Parker, Victor J Hruby, G. Milligan, M. D. Houslay

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)449-457
Number of pages9
JournalBiochemical Journal
Volume268
Issue number2
StatePublished - 1990
Externally publishedYes

Fingerprint

Phosphorylation
Glucagon
Labeling
Hepatocytes
Tetradecanoylphorbol Acetate
8-Bromo Cyclic Adenosine Monophosphate
Immune Sera
Phosphotransferases
Guanine Nucleotides
Vasopressins
Immunoprecipitation
Angiotensin II
Carrier Proteins
Acetates
Phosphoamino Acids
Transducin
Ligands
Phosphoserine
Membranes
Arginine Vasopressin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bushfield, M., Murphy, G. J., Lavan, B. E., Parker, P. J., Hruby, V. J., Milligan, G., & Houslay, M. D. (1990). Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes. Biochemical Journal, 268(2), 449-457.

Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes. / Bushfield, M.; Murphy, G. J.; Lavan, B. E.; Parker, P. J.; Hruby, Victor J; Milligan, G.; Houslay, M. D.

In: Biochemical Journal, Vol. 268, No. 2, 1990, p. 449-457.

Research output: Contribution to journalArticle

Bushfield, M, Murphy, GJ, Lavan, BE, Parker, PJ, Hruby, VJ, Milligan, G & Houslay, MD 1990, 'Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes', Biochemical Journal, vol. 268, no. 2, pp. 449-457.
Bushfield M, Murphy GJ, Lavan BE, Parker PJ, Hruby VJ, Milligan G et al. Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes. Biochemical Journal. 1990;268(2):449-457.
Bushfield, M. ; Murphy, G. J. ; Lavan, B. E. ; Parker, P. J. ; Hruby, Victor J ; Milligan, G. ; Houslay, M. D. / Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes. In: Biochemical Journal. 1990 ; Vol. 268, No. 2. pp. 449-457.
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T1 - Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes

AU - Bushfield, M.

AU - Murphy, G. J.

AU - Lavan, B. E.

AU - Parker, P. J.

AU - Hruby, Victor J

AU - Milligan, G.

AU - Houslay, M. D.

PY - 1990

Y1 - 1990

N2 - 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.

AB - 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.

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