Cytosolic Gα_s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth

It is now evident that Gα_s traffics into cytosol following G protein-coupled receptor activation, and α subunits of some heterotrimeric G-proteins, including Gα_s bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein α subunits remain unclear. In this study, several biochemical approaches demonstrated that activated Gα_s directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Gα_s colocalized with microtubules. The activated, GTP-bound, Gα_s mimicked tubulin in serving as a GTPase activator for β-tubulin. As a result, activated Gα_s made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of Gα_s activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated Gα_s or activation of Gα_s with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Gα_s acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.