A specific domain of Giα required for the transactivation of Giα by tubulin is implicated in the organization of cellular microtubules

Abstract

Gsα, Giα1, and Gqα subunits bind tubulin with high affinity, whereas transducin (Gtα) does not. The interaction between tubulin and Gα, which also involves the direct transfer of GTP from tubulin to Gα (transactivation), is not yet fully understood. This study, using chimeras of Giα and Gtα, showed that the Giα (215-295) segment converted Gtα to bind to tubulin and this chimera (chimera 1) could be transactivated by tubulin. Insertion of Gtα (237-270) into chimera 1 to form chimera 2 resulted in a protein that, like Gtα, did not bind tubulin. Thus, it was thought that the Giα (237-270) domain was essential to modulate the binding of Giα1 to tubulin. Surprisingly, when domain (237-270) of Giα was replaced by Gtα (237-270) to form chimera 3, the chimera bound to tubulin with a similar affinity (KD ≅120 nM) as wild-type Giα1. However, even though chimera 3 displayed normal GTP binding, it was not transactivated by GTP-tubulin. Furthermore, when these chimeras were expressed in COS-1 cells, cellular processes in cells overexpressing Giα1 or chimera 1 were more abundant and longer than those in native cells. Gα was seen throughout the length of the process. Morphology of cells expressing chimera 2 was identical to controls. Consistent with the role of Chimera 3 as a "dominant negative" Gα, cells transfected with chimera 3 had only few truncated processes. This study demonstrates that although Giα (237-270) is not obligatory for the binding of Giα to tubulin, it is crucial for the transactivation of Gα by tubulin. These results also suggest that the transactivation of Gα by tubulin may play an important role in modulating microtubule organization and cell morphology.