title: Distinct phosphoinositide 3-kinases mediate mast cell degranulation in response to G-protein-coupled versus FcepsilonRI receptors.
abstract: Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110beta and p85/p110delta but not p85/p110alpha are required for antigen-mediated calcium flux in RBL-2H3 cells (Smith, A. J., Surviladze, Z., Gaudet, E. A., Backer, J. M., Mitchell, C. A., and Wilson, B. S. et al., (2001) J. Biol. Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110delta and p110beta blocked FcepsilonR1-mediated degranulation in response to FcepsilonRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110alpha, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110alpha mediates a calcium-independent signal during degranulation. In contrast, only p110beta was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110alpha in antigen-stimulated degranulation; (b) a requirement for p110beta in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Windmiller
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D002217:Carbachol / Q000494:pharmacology; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D017455:Receptors, IgE / Q000378:metabolism*
chemical_list: D018679:Cholinergic Agonists; D008024:Ligands; D017455:Receptors, IgE; D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; C496628:PRKCA protein, human; C497258:PRKCD protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D019204:GTP-Binding Proteins