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ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils
A Fensome1, J Whatmore, C Morgan
1Department of Physiology, University College London, London WC1E 6JJ, United Kingdom.
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.
Insights
Formyl-methionyl-leucyl-phenylalanine (fMLP) activates phospholipase D (PLD) in neutrophils via Gi proteins, requiring ADP-ribosylation factor (ARF) and Rho proteins. Phosphoinositide 3-kinase is not essential for this signaling pathway.
Area of Science:
- Cellular signaling pathways
- Immunology
- Biochemistry
Background:
- Neutrophil activation by fMLP stimulates phospholipase D (PLD) through an unidentified signaling cascade.
- Small GTPases, including ADP-ribosylation factor (ARF) and Rho proteins, are known regulators of PLD1 activity.
- Cytosol depletion in neutrophils via streptolysin O permeabilization removes ARF but not Rho proteins, rendering fMLP-stimulated PLD activation refractory.
Purpose of the Study:
- To elucidate the signaling pathway responsible for fMLP-induced activation of neutrophils.
- To determine the specific roles of ARF, Rho proteins, Gi proteins, and phosphoinositide 3-kinase (PI3K) in fMLP-stimulated PLD activation.
Main Methods:
- Utilized cytosol-depleted human neutrophils and reconstituted with myristoylated ARF1 (myr-ARF1).
- Employed C3 toxin to inactivate Rho proteins.
- Investigated the involvement of Gi proteins and PI3K using pertussis toxin and wortmannin, respectively.
- Assessed protein translocation to membranes via Western blotting.
Main Results:
- Readdition of myr-ARF1, but not non-myr-ARF1, restored fMLP-stimulated PLD activity in cytosol-depleted neutrophils.
- C3 toxin reduced ARF-reconstituted PLD activity, indicating Rho protein synergy with ARF.
- Pertussis toxin inhibited PLD activity in both intact and reconstituted cells, highlighting the necessity of Gi2/Gi3 proteins.
- Wortmannin inhibited fMLP-stimulated PLD activity in intact cells but not in myr-ARF1-reconstituted cells, and did not affect ARF/Rho translocation.
Conclusions:
- Activation of Gi proteins is essential for fMLP-induced ARF/Rho activation and subsequent PLD stimulation in human neutrophils.
- Phosphoinositide 3-kinase is not required for fMLP-stimulated PLD activation, despite its role in intact neutrophils.
- ARF and Rho proteins act synergistically, with Gi protein activation being a prerequisite for their activation by fMLP.