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 Gi

FEBS Letters

Pyne, Nigel J.¹  Murphy, Gregory J.¹  Milligan, Graeme¹  Houslay, Miles D.¹ 

[1] Molecular Pharmacology Group, Institute of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland

关键词: Adenylate cyclase;    Guanine nucleotide regulatory protein;    Phosphorylation;    Phorbol ester;    Protein kinase;    Desensitization;    Glucagon;    (Hepatocyte);    p[NH]ppG;    guanylyl 5′-imidodiphosphate;    TPA;    12-O-tetradecanoyl phorbol-13-acetate;    SDS-PAGE;    SDS-polyacrylamide gel electrophoresis;   

DOI  :  10.1016/0014-5793(89)81221-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown