Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with Pasteurella multocida Toxin

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G q , G 12 and G i . PMT has been shown by others to lead to the deamidation of recombinant Gα i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G q and G i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα 11 , a member of the G q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα 13 was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα i , but not Gα q . We also show that PMT inhibits the GTPase activity of G q .