Cytosolic G{alpha}s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth.
It is now evident that Galpha(s) traffics into cytosol following G protein-coupled receptor activation, and alpha subunits of some heterotrimeric G-proteins, including Galpha(s) bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein alpha subunits remain unclear. In this study, several biochemical approaches demonstrated that activated Galpha(s) directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Galpha(s) colocalized with microtubules. The activated, GTP-bound, Galpha(s) mimicked tubulin in serving as a GTPase activator for beta-tubulin. As a result, activated Galpha(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 Galpha(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 Galpha(s) or activation of Galpha(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 Galpha(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.
Yu
Jiang-Zhou
JZ
Departments of Physiology and Biophysics and Psychiatry, University of Illinois, Chicago, Illinois 60612, USA. yujz64@uic.edu
Dave
Rahul H
RH
Allen
John A
JA
Sarma
Tulika
T
Rasenick
Mark M
MM
D000818:Animals; D002772:Cholera Toxin / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D014404:Tubulin / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases