EVIDENCE FOR RHO-MEDIATED AGONIST STIMULATION OF PHOSPHOLIPASE-D IN RAT1 FIBROBLASTS - EFFECTS OF CLOSTRIDIUM-BOTULINUM C3 EXOENZYME

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 botuli num modifies Rho proteins and inhibits their function. When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity st imulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol este r. Neither the time course nor agonist dose response for LPA-stimulate d PLD activity was altered in C3-treated cells. In contrast to the eff ects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-ph ospholipase C activity was not altered in C3-treated cells, Surprising ly, C3 treatment led to a decrease in the amount of RhoA protein, indi cating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins. As described previously, C3 treatmen t led to the inhibition of LPA-stimulated actin filament formation. Ho wever, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity. Interestingly, stimu lation of cells with LPA caused a rapid enrichment of RhoA in the part iculate fraction of cell lysates. These data support an in vivo role f or RhoA in agonist-stimulated PLD activity that is separate from its r ole in actin fiber formation.