ADP-RIBOSYLATION FACTOR PROTEINS MEDIATE AGONIST-INDUCED ACTIVATION OF PHOSPHOLIPASE-D

The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-de rived growth factor (PDGF) and phorbol esters (PMA) has been investiga ted. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridiun botulinu m C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in t he presence of GTP gamma S, promoted the association of ARF and RhoA w ith cell membranes. Cells depleted of ARF and Rho by digitonin permeab ilization showed a significant reduction of the activity of phospholip ase D. Recombinant ARF was sufficient to restore agonist-induced PLD a ctivity to digitonin-permeabilized, cytoplasm-depleted cells. In contr ast, isoprenylated recombinant RhoA had no effects in this reconstitut ion assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-AR F6 had any effects on agonist-dependent PLD activity. However, dominan t-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDG F but only partially inhibited the effects of PMA. These results demon strate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.