ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

Abstract

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD 1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the G i family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARFrestored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for G i2 /G i3 protein. In contrast, wortmannin inhibited only fMLPstimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLPstimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of G i proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.

Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis

“…Within 5-10 min a substantial proportion of ARF1 proteins was found in the supernatant. In contrast, Rho proteins did not substantially leak out of cells, similar to results obtain previously with human neutrophils [16].…”

“…Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned [13,14]. Regulation of PLD1 has been intensively studied and is known to be directly and synergistically regulated by ARF1, Rho proteins (RhoA, Rac and Cdc42) and protein kinase Cα [15,16].…”

Leukocyte Microparticles Stimulate Endothelial Cell Cytokine Release and Tissue Factor Induction in a JNK1 Signaling Pathway

“…1), and in agreement with previous observations (16). Pretreatment with 100 ng/ml pertussis toxin (22) did not reduce MP release from fMLP-or IL-8-stimulated PMN suspensions (Fig. 1).…”

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

“…We have previously demonstrated that ARF translocation correlated with fMLP-induced PLD activation in HL60 granulocytes [15]. More recently, a concomitant PLD activation and rapid enrichment of RhoA in the particulate fraction was documented in LPAstimulated Rat1 fibroblasts [29] and fMLP-stimulated neutrophils [16]. Despite these advances, many aspects of the mechanisms linking receptor occupation to PLD activation remain largely unknown.…”

“…We have previously shown that ARF translocated to the membrane fraction of HL60 cells in response to fMLP and that this translocation correlated with an increase in the GTP␥S-stimulated membranebound PLD activity [15]. Phorbol 12-myristate 13-acetate (PMA), a non-physiological PKC agonist, had a similar effect on membrane-bound PLD activity and ARF translocation [15].fMLP in neutrophils [16] and that lysophosphatidic acid (LPA) stimulation induces the enrichment of RhoA in the particulate fraction of Rat1 fibroblasts [17].…”