TY - JOUR
T1 - Distinct phosphoinositide 3-kinases mediate mast cell degranulation in response to G-protein-coupled versus FcεRI receptors
AU - Windmiller, David A.
AU - Backer, Jonathan M.
PY - 2003/4/4
Y1 - 2003/4/4
N2 - Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110β and p85/p110δ but not p85/p110α 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 p110δ and p110β blocked FcεR1-mediated degranulation in response to FcεRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110α, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110α mediates a calcium-independent signal during degranulation. In contrast, only p110β 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 p110α in antigen-stimulated degranulation; (b) a requirement for p110β 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.
AB - Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110β and p85/p110δ but not p85/p110α 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 p110δ and p110β blocked FcεR1-mediated degranulation in response to FcεRI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110α, despite the fact that these antibodies have no effect on antigen-induced calcium flux. These data suggest that p110α mediates a calcium-independent signal during degranulation. In contrast, only p110β 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 p110α in antigen-stimulated degranulation; (b) a requirement for p110β 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.
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U2 - 10.1074/jbc.M211787200
DO - 10.1074/jbc.M211787200
M3 - Article
C2 - 12529321
AN - SCOPUS:0038823621
SN - 0021-9258
VL - 278
SP - 11874
EP - 11878
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -