ADP-ribosylation of G alpha i and G alpha o in pituitary cells enhances their recognition by antibodies directed against their carboxyl termini

J Recept Signal Transduct Res. May-Jul 1996;16(3-4):169-90. doi: 10.3109/10799899609039947.

Abstract

Using antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of G alpha s, G alpha i1,2, G alpha i3, G alpha o2, and G beta subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of G alpha s and this decrease was kinetically coupled to the rate of G alpha s ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of G alpha i and G alpha o enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the G alpha carboxyl termini. Such enhancement was not observed when antibodies directed against the NH2-termini were used. These findings are consistent with the fact that ADP-ribosylation by IAP occurs on the cysteine located in the carboxyl terminal part of G alpha i and G alpha o. These observations mean that the kinetics and extent of Gi and Go ADP-ribosylation by IAP in whole pituitary cells and membrane preparations can be followed. It could be that ADP-ribosylation causes conformational changes in G alpha i and G alpha o. Indeed, we observed that ADP-ribosylated G alpha i was more sensitive to trypsin proteolysis and that the ADP-ribosylation rates of G alpha i and G alpha o in whole cells were comparable to the rate of loss of coupling between inhibitory neurohormone receptors and adenylyl cyclase.

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism*
  • Adenylate Cyclase Toxin
  • Adenylyl Cyclases / metabolism
  • Animals
  • Cells, Cultured
  • Female
  • GTP-Binding Proteins / analysis*
  • GTP-Binding Proteins / immunology
  • GTP-Binding Proteins / metabolism
  • Immunoblotting
  • Pertussis Toxin
  • Pituitary Gland / chemistry*
  • Rats
  • Rats, Sprague-Dawley
  • Trypsin / pharmacology
  • Virulence Factors, Bordetella / pharmacology

Substances

  • Adenylate Cyclase Toxin
  • Virulence Factors, Bordetella
  • Adenosine Diphosphate Ribose
  • Pertussis Toxin
  • Trypsin
  • GTP-Binding Proteins
  • Adenylyl Cyclases