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

N J Pyne, G J Murphy, G Milligan, M D Houslay

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61 Citations (Scopus)

Abstract

The antiserum AS7 can specifically immunoprecipitate alpha-Gi from membrane extracts as well as from a mixture of purified alpha-Gi and alpha-Go as ascertained using [32P]ADP-ribosylated G-proteins. Using this antiserum to immunoprecipitate alpha-Gi from hepatocytes labelled with 32P it was evident that alpha-Gi 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 alpha-Gi. This was accompanied by the loss of inhibitory effect of Gi 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 alpha-Gi in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of alpha-Gi in intact hepatocytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves alpha-Gi in its holomeric state.
Original languageEnglish
Pages (from-to)77-82
Number of pages6
JournalFEBS Letters
Volume243
Issue number1
DOIs
Publication statusPublished - 1989

Keywords

  • adenylate cyclase
  • Animals
  • antigen-antibody complex
  • cells
  • GTP-binding proteins
  • glucagon
  • immune sera
  • kinetics
  • Llver
  • phosphorylation
  • rats

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