ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils

Related Experiment Video

Updated: Oct 29, 2026

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration
10:43

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Published on: May 19, 2016

A Fensome1, J Whatmore, C Morgan

  • 1Department of Physiology, University College London, London WC1E 6JJ, United Kingdom.

The Journal of Biological Chemistry
|May 28, 1998
PubMed

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.

More Related Videos

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes08:49

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Published on: March 14, 2021

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation12:49

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Published on: March 21, 2020

Related Concept Videos

IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...
Mechanism of Filopodia Formation01:39

Mechanism of Filopodia Formation

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Mechanism of Lamellipodia Formation01:31

Mechanism of Lamellipodia Formation

Cells migrating in response to external stimuli form lamellipodia, which are thin membrane protrusions supported by a mesh of linked, branched, or unbranched actin filaments. These actin filaments interact with myosin motor proteins, creating the dynamic actomyosin complex within the cytoskeleton. Contractility, or the ability to generate contractile stress, is inherent to the actomyosin complex. It helps cells detect the stiffness of the surrounding ECM and exert contractile force for...
Cell Polarization by Rho Proteins01:21

Cell Polarization by Rho Proteins

Cell polarity is the asymmetric distribution of cellular and membrane components, making one side of the cell different from the other. This polarity is essential to many processes such as embryogenesis, axon migration, glucose transport across epithelial cells, and directional cell migration. A migrating cell responds to intracellular or extracellular signals via molecular cascades that reorganize the actin cytoskeleton to establish this polarity. In these cells, the Rho family proteins Cdc42,...
Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...