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An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

ABSTRACT: Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation.

SUBMITTER: Gandhi PN

PROVIDER: S-EPMC1223982 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form. RESULTS: We report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response. CONCLUSION: T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.

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An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

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