ADP-ribosylation of G alpha i and G alpha o in pituitary cells enhances their recognition by antibodies directed against their carboxyl termini.
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.
Cussac
D
D
ICNE UMR 9941 CNRS, Faculté de Médecine Secteur Nord, Marseille.
Kordon
C
C
Enjalbert
A
A
Saltarelli
D
D
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D014357:Trypsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases