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ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

Pubmed ID: 12799371


  • Mitchell R
  • Robertson DN
  • Holland PJ
  • Collins D
  • Lutz EM
  • Johnson MS


The Journal of biological chemistry

Publication Data

September 5, 2003

Associated Grants


Mesh Terms

  • ADP-Ribosylation Factor 1
  • ADP-Ribosylation Factors
  • Animals
  • Biotinylation
  • Blotting, Western
  • Brefeldin A
  • COS Cells
  • Carbachol
  • Cell Line
  • Cell Membrane
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Enzyme Inhibitors
  • Epitopes
  • Estrenes
  • Glutathione Transferase
  • Humans
  • Immunoblotting
  • Inhibitory Concentration 50
  • Ligands
  • Models, Biological
  • Mutation
  • Phospholipase D
  • Precipitin Tests
  • Protein Binding
  • Protein Kinase C
  • Protein Structure, Tertiary
  • Protein Transport
  • Pyrrolidinones
  • Receptor, Muscarinic M3
  • Receptors, Muscarinic
  • Signal Transduction
  • Subcellular Fractions
  • Time Factors
  • Transfection
  • Tumor Cells, Cultured