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

Research output: Contribution to journalArticle

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.
LanguageEnglish
Pages77-82
Number of pages6
JournalFEBS Letters
Volume243
Issue number1
DOIs
Publication statusPublished - 1989

Fingerprint

Phosphorylation
Phorbol Esters
Glucagon
GTP-Binding Proteins
Hepatocytes
Adenylyl Cyclases
Adenosine Diphosphate
Pertussis Toxin
Immune Sera
Membranes
Colforsin
Cyclic AMP
NAD
Protein Kinase C
Tumors
Insulin
Neoplasms

Keywords

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

Cite this

@article{5b20bafe0b9a409c8860fcd77dafbde6,
title = "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",
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.",
keywords = "adenylate cyclase, Animals, antigen-antibody complex, cells, GTP-binding proteins, glucagon, immune sera, kinetics, Llver, phosphorylation, rats",
author = "Pyne, {N J} and Murphy, {G J} and G Milligan and Houslay, {M D}",
year = "1989",
doi = "10.1016/0014-5793(89)81221-9",
language = "English",
volume = "243",
pages = "77--82",
journal = "FEBS Letters",
issn = "0014-5793",
number = "1",

}

TY - JOUR

T1 - 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

AU - Pyne, N J

AU - Murphy, G J

AU - Milligan, G

AU - Houslay, M D

PY - 1989

Y1 - 1989

N2 - 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.

AB - 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.

KW - adenylate cyclase

KW - Animals

KW - antigen-antibody complex

KW - cells

KW - GTP-binding proteins

KW - glucagon

KW - immune sera

KW - kinetics

KW - Llver

KW - phosphorylation

KW - rats

U2 - 10.1016/0014-5793(89)81221-9

DO - 10.1016/0014-5793(89)81221-9

M3 - Article

VL - 243

SP - 77

EP - 82

JO - FEBS Letters

T2 - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 1

ER -