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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 PLD1 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 Gi 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, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.
Highlights
We conclude that the activation of phospholipase D (PLD) by the fMLP receptor is dependent on receptor-activated ADP-ribosylation factor (ARF) and Rho proteins in human neutrophils coupled via Gi proteins and is not obligatorily dependent on PI 3-kinase activation
ARF Restores fMLP-dependent PLD Activity in Cytosol-depleted Human Neutrophils—To examine a requirement for ARF proteins in fMLP-stimulated PLD activity, human neutrophils were permeabilized with streptolysin O for 10 min to deplete the cells of their freely diffusable cytosolic proteins
This loss is coincident with the inability of fMLP to stimulate PLD activity in cytosol-depleted cells (Ref. 26 and Fig. 1B). fMLP regains the ability to stimulate PLD activity provided that myr-ARF1 is added to the permeabilized cells (Fig. 1B)
Summary
Activation of PLD in intact neutrophils is inhibited by wortmannin, a relatively selective inhibitor of PI 3-kinase, wortmannin treatment is not inhibitory to the fMLP-stimulated recruitment of ARF and Rho to membranes. Both myristoylated (myrARF1) and nonmyristoylated (ARF1) ARF proteins were examined for their ability to restore fMLP-dependent PLD activity in cytosol-depleted neutrophils. Pertussis toxin pretreatment inhibits fMLP-stimulated PLD activity when the agonist and the permeabilizing agent streptolysin O are added simultaneously, conditions in which the cytosolic proteins are still present (Fig. 3B).
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