The small GTPases of the Rho family play a key role in a number of signaling pathways activated by lysophosphatidic acid (LPA). However, little is known concerning the mechanism of regulation of these proteins. In this study we demonstrate that in Swiss 3T3 fibroblasts, LPA induces a sustained, time-dependent relocalization of RhoA to the Triton X-100-soluble low speed membrane fraction, which can be reversed by removal of LPA from the medium. Translocation was only observed with micromolar concentrations of LPA and was inhibited by pretreating the cells with pertussis toxin but not with tyrosine kinase inhibitors. LPA also induced translocation of CDC42Hs to the membranes but had no effect on the distribution of Rac1, RhoB, or Rho-GDI. Translocation of RhoA was also induced by endothelin-1. Conversely, platelet-derived growth factor did not cause the translocation of RhoA to any membrane fraction but stimulated relocalization of Rac1 to the high speed membrane fraction. Significantly, incubation of cell lysates with guanosine 5-O-(thiotriphosphate) was sufficient to translocate RhoA, Rac1, and CDC42Hs from the cytosol to the membranes, whereas incubation with GDP had the opposite effect. These data suggest that the translocation of the Rho family proteins to the membrane fraction is controlled by their activation state and that agonists show selectivity in inducing the activation/translocation of these proteins.The Rho family of Ras-related small GTPases, which includes RhoA, -B, -C, and -G, Rac1 and -2, and CDC42Hs and TC10 (1), plays an important role in the regulation of cell function. Rho is involved in the formation of actin stress fibers and focal adhesions (2-4) and in the motile response of cells (3). Rac is important in the NADPH oxidase-mediated phagocytic response in leukocytes (5) and in actin polymerization associated with membrane ruffling and lamellipodia formation in fibroblasts (4, 6). CDC42Hs is involved in the formation of filopodia in fibroblasts (4), and its yeast homologue regulates bud site assembly in Saccharomyces cerevisiae (7).The proteins of the Rho family cycle between GTP-bound (active) and GDP-bound (inactive) states, aided by a number of regulatory proteins. Several guanine nucleotide exchange factors, which promote binding of GTP to Rho family members by facilitating the release of GDP, have recently been identified (8). A large number of GTPase-activating proteins, which act on Rho proteins, have also been characterized (9). However, most of the exchange factors or GTPase-activating proteins identified thus far do not show absolute specificity for one member of the Rho family, although some show preferences for particular G proteins. In addition, all Rho family members bind to a cytosolic regulatory protein, Rho GDP dissociation inhibitor (Rho-GDI), 1 which inhibits GDP dissociation (10) and GTP hydrolysis (11) and may keep the GTPases localized mainly in the cytosolic compartment (12).A number of signaling pathways in fibroblasts that are activated by the bioactive lipid lysop...
Small GTP-binding proteins of the Rho family are implicated in the in vitro regulation of phosphatidylcholine hydrolysis by phospholipase D (PLD). However, their role in agonist-stimulated PLD activity in whole cells is not clear. The ribosyltransferase C3 from Clostridium botulinum modifies Rho proteins and inhibits their function. When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity stimulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol ester. Neither the time course nor agonist dose response for LPA-stimulated PLD activity was altered in C3-treated cells. In contrast to the effects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-phospholipase C activity was not altered in C3-treated cells. Surprisingly, C3 treatment led to a decrease in the amount of RhoA protein, indicating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins. As described previously, C3 treatment led to the inhibition of LPA-stimulated actin filament formation. However, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity. Interestingly, stimulation of cells with LPA caused a rapid enrichment of RhoA in the particulate fraction of cell lysates. These data support an in vivo role for RhoA in agonist-stimulated PLD activity that is separate from its role in actin fiber formation.
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