ADP-RIBOSYLATION FACTOR AND RHO-PROTEINS MEDIATE FMLP-DEPENDENT ACTIVATION OF PHOSPHOLIPASE-D IN HUMAN NEUTROPHILS

Activation of intact human neutrophils by fMLP stimulates phospholipas e D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribos ylation factor (ARF), and Rho proteins regulate the activity of PLD1 d irectly. Cell permeabilization with streptolysin O leads Do loss of cy tosolic proteins including ARF but not Rho proteins from the human neu trophils, PLD activation by fMLP is refractory in these cytosol-deplet ed cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-st imulated PLD activity, C3 toxin, which inactivates Rho proteins, reduc es the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF, To iden tify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotr imeric G proteins of the G(i) family and for phosphoinositide 3-kinase , respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, i ndicating a requirement for G(i2)/G(i3) protein. In contrast, wortmann in inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulat ed translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin, It is concluded that activation of G(i) proteins is ob ligatory for ARF/Rho activation by fMLP, but activation of phosphoinos itide 3-kinase is not required.