Localizing Rhos

Science's STKE  16 Jan 2001:
Vol. 2001, Issue 65, pp. tw4
DOI: 10.1126/stke.2001.65.tw4

Members of the Rho family of guanosine triphosphatases (GTPases) regulate several signal transduction pathways. More than 15 members of this family have been identified, and cells typically express more than one family member. One possible mechanism by which signal specificity is conveyed is through different subcellular localizations of the various Rho proteins. Michaelson et al. used green fluorescent protein (GFP) fused to each of seven members of the Rho family to determine the cellular localization of these proteins. They also showed that a hypervariable region in the COOH-terminus and differences in posttranslation lipid modifications were responsible for the differences in localization. The localizations were confirmed with immunocytochemical analysis of endogenous Rho proteins. Coexpression of oncogenic Dbl (a Rho guanine nucleotide exchange factor) to stimulate GFP-pCdc42hs, GFP-bCdc42hs, or GFP-RhoA led to redistribution of these proteins from the cytosol to lamellipodia membranes. The ability of Rho members to interact with the negative regulator of GTPase activity RhoGDI (GDP dissociation inhibitor) was measured by the ability of coexpressed RhoGDI to sequester the GFP-Rho fusion protein in the cytosol. Interaction with RhoGDI could be regulated by type of lipid modification and could be inhibited by palmitoylation of RhoA. Finally, this in vivo assay of RhoGDI binding was used to test whether dominant negative or constitutively active forms of the GTPases were competent to interact with the inhibitor. These results were confirmed by coimmunoprecipitation assays. It was surprising that not all dominant negative forms or constitutively activated forms exhibited the predicted interaction or lack thereof, respectively, with RhoGDI. Sequestration of mutants through interaction with RhoGDI may complicate interpretation of experiment monitoring the effects of such altered forms of the Rho proteins.

D. Michaelson, J. Silletti, G. Murphy, P. D'Eustachio, M. Rush, M. R. Phillips, Differential localization of Rho GTPases in live cells: Regulation by hypervariable regions and RhoGDI binding. J. Cell Biol. 152, 111-126 (2001). [Abstract] [Full Text]