ADP-Ribosylation of Gαi and Gαo in Pituitary Cells Enhances their Recognition by Antibodies Directed Against their Carboxyl Termini

Abstract

Using antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of Gαs, Gαil,2, Gαi3, Gαo2, and Gβ subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of Gαs and this decrease was kinetically coupled to the rate of Gás ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of Gαi and Gαo enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the Gα 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 Gαi and Gαo. 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 Gαi and GαO. Indeed, we observed that ADP-ribosylated Gαi was more sensitive to trypsin proteolysis and that the ADP-ribosylation rates of Gαi and Gαo in whole cells were comparable to the rate of loss of coupling between inhibitory neurohormone receptors and adenylyl cyclase.