Distinct Phosphoinositide 3-Kinases Mediate Mast Cell Degranulation in Response to G-protein-coupled VersusFcεRI Receptors
2003; Elsevier BV; Volume: 278; Issue: 14 Linguagem: Inglês
10.1074/jbc.m211787200
ISSN1083-351X
AutoresDavid A. Windmiller, Jonathan Backer,
Tópico(s)Asthma and respiratory diseases
ResumoPhosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110ॆ 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 p110ॆ 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 p110ॆ 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 p110ॆ 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. Phosphoinositide (PI) 3-kinases are critical regulators of mast cell degranulation. The Class IA PI 3-kinases p85/p110ॆ 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 p110ॆ 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 p110ॆ 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 p110ॆ 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. G-protein-coupled receptor dinitrophenol enhanced green fluorescent protein fluorescein isothyocyanate Fab1/YOTB/Vac1p/EEA1-homology domain phosphatidylinositol 3-phosphate phospholipase C protein kinase C Phox homology domain regulator of G-protein signaling phosphoinositide Hanks' basic salt solution Mast cells are important cellular mediators of allergic responses in humans (1Forsythe P. Ennis M. Inflamm. Res. 1999; 48: 301-307Google Scholar). Moreover, increased levels of mast cells and mast cell-derived inflammatory mediators are found in brochoalveolar lavage fluid from asthmatics, suggesting a role for mast cells in the etiology of clinical asthma (2Olsson N. Rak S. Nilsson G. J. Allergy Clin. Immunol. 2000; 105: 455-461Google Scholar, 3Gibson P.G. Saltos N. Borgas T. J. Allergy Clin. Immunol. 2000; 105: 752-759Google Scholar, 4Bingham C.O. Austen K.F. J. Allergy Clin. Immunol. 2000; 105: S527-S534Google Scholar). Cross-linking of cell surface FcεRI receptors leads to the release of pre-formed mediators present in mast cell granules, as well as the induction of cytokines and bioactive lipids (5Reischl I.G. Coward W.R. Church M.K. Biochem. Pharmacol. 1999; 58: 1841-1850Google Scholar). Release of these inflammatory molecules in the lung is likely to contribute to inflammation and vasoconstriction during asthma. Antigen-mediated degranulation is enhanced by co-stimulation of mast cells with adenosine, which is an important contributor to airway inflammation in asthma (6Holgate S.T. Church M.K. Polosa R. Ann. N. Y. Acad. Sci. 1991; 629: 227-236Google Scholar). The initial signaling events during antigen-stimulated degranulation have been well studied. FcεRI cross-linking leads to recruitment and activation of lyn and syk tyrosine kinases, with subsequent phosphorylation of tyrosine residues in the FcεRI γ-chain (5Reischl I.G. Coward W.R. Church M.K. Biochem. Pharmacol. 1999; 58: 1841-1850Google Scholar, 7Turner H. Kinet J.P. Nature. 1999; 402: B24-B30Google Scholar). This leads to the recruitment, phosphorylation and activation of phospholipase Cγ, and generation of inositol trisphosphate and diacylglycerol from the hydrolysis of plasma membrane phosphatidylinositol (4,5)-bisphosphate. Inositol trisphosphate-mediated release of intracellular calcium stores and activation of classical and novel isoforms of protein kinase C (8Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Google Scholar) are required for the opening of plasma membrane calcium channels. The increase in intracellular calcium levels is critical for degranulation, as evidenced by the fact that thapsigargin or calcium ionophores can induce mast cell degranulation in the absence of additional stimuli. G-protein-coupled receptors (GPCRs)1 also regulate mast cell degranulation. In bone marrow-derived mast cells and in a cell culture model, the RBL-2H3 basoleukemic line, adenosine can synergistically enhance degranulation in response to FceRI crosslinking, although it is not sufficient to stimulate degranulation (6Holgate S.T. Church M.K. Polosa R. Ann. N. Y. Acad. Sci. 1991; 629: 227-236Google Scholar, 9Ramkumar V. Stiles G.L. Beaven M.A. Ali H. J. Biol. Chem. 1993; 268: 16887-16890Google Scholar, 10Marquardt D.L. Parker C.W. Sullivan T.J. J. Immunol. 1978; 120: 871-878Google Scholar). Adenosine signaling in RBL-2H3 cells is primarily mediated by the A3 adenosine receptor, a Gαi-coupled GPCR (9Ramkumar V. Stiles G.L. Beaven M.A. Ali H. J. Biol. Chem. 1993; 268: 16887-16890Google Scholar). In addition, Beaven and colleagues (11Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Google Scholar) demonstrated that stable expression in RBL-2H3 cells of a heterologous GPCR, the M1 muscarinic acetylcholine receptor, leads to carbachol-stimulated degranulation. Carbachol versus antigen stimulation of RBL-2H3-M1 cells lead to similar changes in calcium mobilization and activation of Erk and phospholipase A2 (12Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402Google Scholar, 13Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595Google Scholar, 14Offermanns S. Jones S.V. Bombien E. Schultz G. J. Immunol. 1994; 152: 250-261Google Scholar), although carbachol-stimulated degranulation used PLCॆ rather than PLCγ to trigger PKC activation and calcium flux (13Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595Google Scholar). The phosphoinositide 3-kinase inhibitor wortmannin is a potent inhibitor of mast cell degranulation (15Kitani S. Teshima R. Morita Y. Ito K. Matsuda Y. Nonomura Y. Biochem. Biophys. Res. Commun. 1992; 183: 48-54Google Scholar), and deletion of the gene for the phosphatidylinositol trisphosphate-phosphatase SHIP markedly enhances mast cell degranulation (16Huber M. Helgason C.D. Damen J.E. Liu L. Humphries R.K. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11330-11335Google Scholar). These data demonstrate that PI 3-kinases are important regulators of antigen-stimulated degranulation. Microinjection of isoform-specific inhibitory antibodies to p110ॆ and p110δ reduce antigen-stimulated calcium flux and membrane ruffling in RBL-2H3 cells (17Smith A.J. Surviladze Z. Gaudet E.A. Backer J.M. Mitchell C.A. Wilson B.S. J. Biol. Chem. 2001; 276: 17213-17220Google Scholar). In addition, recent studies on a mouse knockout of the Class IB PI 3-kinase, p110γ, suggest that p110γ is important in both FceRI and adenosine-mediated degranulation (18Laffargue M. Calvez R. Finan P. Trifilieff A. Barbier M. Altruda F. Hirsch E. Wymann M.P. Immunity. 2002; 16: 441-451Google Scholar). However, the role of Class IA and Class III PI 3-kinases in degranulation itself has not been directly examined. In this paper, we use specific inhibitory antibodies against Class IA and Class III PI 3-kinases to test the requirement for these enzymes during degranulation. Using a single-cell degranulation assay in RBL-2H3 cells, we show that the three Class IA PI 3-kinases are all required for optimal antigen-stimulated degranulation. In contrast, synergistic enhancement of antigen-stimulated degranulation by adenosine specifically requires p85/p110ॆ. Surprisingly, carbachol-stimulated degranulation in RBL-2H3-M1 cells does not require Class I PI 3-kinases and instead requires the Class III enzyme hVPS34. The utilization of PKC isozymes in antigen versus carbachol-stimulated degranulation was also different. These data suggest a novel role of hVPS34 in GPCR signaling and highlight the regulatory complexity of ligand-stimulated degranulation in mast cells. RBL-2H3 and RBL-2H3 cells expressing the M1 muscarinic receptor (RBL-2H3-M1) were cultured in Iscove's modified Dulbecco's medium containing 157 fetal bovine serum on Nunc tissue culture dishes or fibronectin-coated coverslips. Isoform-specific inhibitory antibodies against p110α, p110ॆ, p110δ, and hVPS34 have been described previously (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar, 20Hill K. Welti S. Yu J.H. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Google Scholar, 21Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Google Scholar). All antibodies were affinity-purified and concentrated to 4 mg/ml in phosphate-buffered saline. The eGFP-2X-FYVE construct (22Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Google Scholar) was obtained from Dr Harald Stenmark, The Norwegian Radium Hospital, Norway. Wortmannin was obtained from Sigma, and rottlerin and Go6976 were obtained from Calbiochem. Cells were microinjected using an Eppendorf 5171/5242 semi-automatic micromanipulator/microinjector as described previously (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar). Cells were allowed to recover for 2 h after injection prior to stimulation as described below. RBL-2H3 cells were incubated overnight in 0.1 ॖg/ml anti-DNP IgG. The cells were washed in Hanks' basic salt solution (HBSS) and stimulated for 45 min at 37 °C with HBSS, 1 mm calcium containing 10 ng/ml DNP-albumin. For adenosine experiments, cells were stimulated with 0.5 ng/ml DNP-albumin in the absence or presence of 10 ॖm adenosine or carrier. Alternatively, RBL-2H3-M1 cells were stimulated for 45 min at 37 °C with HBSS containing 100 ॖm carbachol or carrier. In each case, the supernatant was removed and brought to 100 mmcitrate, pH 4.5, 1 mm4-methylumbelliferyl-N-acetyl glucosamine (Sigma). After 15 min at 37 °C, the reaction was stopped with 1/10 volume of 200 mm Na2CO3, glycine, pH 10.7, and substrate hydrolysis measured using a fluorescence spectrophotometer (360 excitation/465 emission). For detection of degranulated cells using annexin V, cells grown on fibronectin-coated coverslips were preloaded as described above, washed in HBSS, and stimulated as described with the additional presence of a 1:10 dilution of Alexa 594 or Alexa 488 annexin V reagent (Molecular Probes, Eugene, OR). The cells were washed, fixed in 3.77 formaldehyde for 10 min at 22 °C, and mounted. Cells were scored for annexin V staining using a Nikon Eclipse 400 upright microscope with a 60 × 1.4 N.A. plan-apo infinity-corrected objective. Each measurement reflects ∼100 injected cells per condition, and the data are the mean from three to five separate experiments. When indicated, cells were transfected with LipofectAMINE Plus according to manufacturer's instructions (Invitrogen). Images were acquired using a Cohu 4910 B/W CCD camera with NIH Image 1.62 analysis software. We and others (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar, 20Hill K. Welti S. Yu J.H. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Google Scholar, 21Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Google Scholar) have previously characterized specific inhibitory antibodies to Class I and Class III PI 3-kinases. To use these reagents to study mast cell degranulation in single cells, we modified a flow cytometry assay developed by Demo et al. (23Demo S.D. Masuda E. Rossi A.B. Throndset B.T. Gerard A.L. Chan E.H. Armstrong R.J. Fox B.P. Lorens J.B. Payan D.G. Scheller R.H. Fisher J.M. Cytometry. 1999; 36: 340-348Google Scholar), in which the binding of fluorescently labeled annexin V is used to identify degranulated cells. As shown in Fig. 1, quiescent anti-DNP IgE-loaded RBL-2H3 cells show no staining with FITC-annexin V (Fig.1B). In contrast, after stimulation of FcεRI receptors with DNP-albumin for 45 min, a clear increase in FITC-annexin V staining is seen in most cells (Fig. 1D). To validate the use of annexin V staining as a single-cell assay, we used the percentage of FITC-annexin V-positive cells as a measure of degranulation, and compared it witho degranulation as determined by a biochemical assay of degranulation, the release of ॆ-hexosaminidase activity. Both assays showed a similar dose response for degranulation in DNP-albumin-stimulated cells (Fig. 1E). In addition, both assays showed similar inhibition by the PI 3-kinase inhibitor wortmannin (Fig. 1F) and by the pan-PKC inhibitor Bisindolmaleimide (data not shown). Thus, the annexin V-based assay accurately reflects ligand-stimulated degranulation of RBL-2H3 cells. It was previously shown that specific inhibition of two isoforms of Class IA PI 3-kinase, p85/p110ॆ and p85/p110δ, diminishes antigen-stimulated calcium flux and membrane ruffling in RBL-2H3 cells (17Smith A.J. Surviladze Z. Gaudet E.A. Backer J.M. Mitchell C.A. Wilson B.S. J. Biol. Chem. 2001; 276: 17213-17220Google Scholar). To directly examine their role in degranulation, anti-DNP IgE-loaded RBL-2H3 cells were microinjected with inhibitory antibodies to p110α, p110ॆ, p110δ, and the Class III PI 3-kinase hVPS34. After a recovery period, the cells were stimulated with 10 ng/ml DNP-albumin for 45 min. Whereas microinjection of rabbit IgG or anti-hVPS34 antibodies had no effects on degranulation, microinjection of antibodies against p110α, p110ॆ, and p110γ inhibited degranulation by 707, 607, and 457, respectively (Fig.2A). We also examined the role of these PI 3-kinases in signaling by the adenosine receptor, a physiologically important contributor to airway inflammation and mast cell activation in asthma (6Holgate S.T. Church M.K. Polosa R. Ann. N. Y. Acad. Sci. 1991; 629: 227-236Google Scholar). Adenosine is not sufficient to induce degranulation but can enhance FcεRI-mediated degranulation in RBL-2H3 cells (9Ramkumar V. Stiles G.L. Beaven M.A. Ali H. J. Biol. Chem. 1993; 268: 16887-16890Google Scholar). Anti-DNP IgE-loaded RBL-2H3 cells were microinjected as above and then stimulated with a suboptimal does of DNP-albumin (0.5 ng/ml) in the absence or presence of 10 ॖm adenosine. In control cells, adenosine caused a 3–4-fold increase in the number of degranulated cells. This was unaffected by microinjection of antibodies against p110α, p110δ, or hVPS34 (Fig. 2B). In contrast, the effects of adenosine were markedly attenuated by antibodies against p110ॆ. These data demonstrate a general requirement for Class IA PI 3-kinase during FcεRI-mediated degranulation and a specific requirement for p85/p110ॆ during adenosine-stimulated degranulation. The RBL-2H3-M1 line, which expresses the M1 acetylcholine receptor, was developed as a model system to study cholinergic regulation of secretion and neurotransmitter release (11Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Google Scholar). Carbachol elicits a robust degranulation response in these cells. To examine the role of PI 3-kinases in M1 muscarinic receptor signaling, we microinjected RBL-2H3-M1 cells with inhibitory anti-PI 3-kinase antibodies and stimulated the cells with 100 ॖm carbachol for 45 min. Unlike antigen-stimulated degranulation, carbachol-stimulated degranulation was unaffected by inhibition of any of the Class I PI 3-kinases (Fig.3A). Surprisingly, carbachol-stimulated degranulation was markedly reduced by inhibition of the Class III enzyme, hVPS34 (Fig. 3A). To confirm the role of hVPS34 in carbachol-stimulated degranulation, we transiently transfected RBL-2H3 or RBL-2H3-M1 cells with an eGFP-linked construct containing two FYVE domains from hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (22Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Google Scholar). Although this construct is useful as a marker for PI(3)P-containing membranes when expressed at low levels, high level overexpression of FYVE domain-containing proteins has been shown to disrupt PI(3)P-mediated signaling, presumably by titration of PI(3)P (24Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Google Scholar). We therefore used high level expression of the construct to disrupt PI(3)P-dependent signaling. We found that overexpression of the eGFP-FYVE in mast cells had no effect on FcεRI-mediated degranulation in RBL-2H3 cells, but it inhibited carbachol-stimulated degranulation in the RBL-2H3-M1 cells (Fig. 3B). Thus, both hVPS34 and its lipid product, PI(3)P, are required for carbachol-stimulated degranulation. To further characterize differential signaling during antigen versus carbachol-stimulated degranulation, we examined the requirement for PKCॆ and PKCδ. These isoforms have previously been shown to be stimulatory for degranulation in RBL-2H3 cells in response to antigen (8Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Google Scholar). We found that the PKCδ inhibitor rottlerin had similar inhibitory effects on both antigen and carbachol-stimulated degranulation, with IC50values of ∼3–10 ॖm in the two cell lines (Fig.4B). In contrast, the PKCα/ॆ inhibitor Go6976 potently inhibited antigen-stimulated degranulation in RBL-2H3 cells (IC50 10 nm), but had no effect on carbachol-stimulated degranulation in the RBL-2H3-M1 cells (Fig. 4A). These data show that the utilization of both PKC and PI 3-kinase isoforms is different in carbacholversus antigen-stimulated degranulation. In this report we have used well characterized isoform-specific anti-PI 3-kinase antibodies (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar, 20Hill K. Welti S. Yu J.H. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Google Scholar, 21Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Google Scholar), in conjunction with a single-cell assay for degranulation, to identify the roles of distinct PI 3-kinases during mast cell degranulation. The assay is based on a previously published flow cytometry assay (23Demo S.D. Masuda E. Rossi A.B. Throndset B.T. Gerard A.L. Chan E.H. Armstrong R.J. Fox B.P. Lorens J.B. Payan D.G. Scheller R.H. Fisher J.M. Cytometry. 1999; 36: 340-348Google Scholar), which used fluorescently labeled annexin V to quantitate the increase in exofacial phosphatidylserine that occurs in the plasma membrane of degranulated cells. A similar increase in exofacial phosphatidylserine occurs in apoptotic cells, and fluorescent annexin V is a commonly used assay for apoptosis (25Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Google Scholar). In the single cell assay presented here, degranulation is expressed as the percentage of annexin V-positive cells per field. The dose-response curve for antigen-stimulated degranulation using this assay correlates well with biochemical assays for degranulation and shows similar sensitivity to wortmannin. This assay provides a useful adjunct to previous single-cell studies in RBL-2H3 cells, which focused on calcium flux and membrane ruffling (17Smith A.J. Surviladze Z. Gaudet E.A. Backer J.M. Mitchell C.A. Wilson B.S. J. Biol. Chem. 2001; 276: 17213-17220Google Scholar). The importance of examining degranulation itself, as opposed to degranulation-related events, is shown in the experiments on the role of Class IA PI 3-kinases during antigen-stimulated degranulation. Antigen-stimulated calcium flux has previously been shown to require p85/p110ॆ and p85/p110δ but not p85/p110α (17Smith A.J. Surviladze Z. Gaudet E.A. Backer J.M. Mitchell C.A. Wilson B.S. J. Biol. Chem. 2001; 276: 17213-17220Google Scholar). The role of these enzymes in calcium flux is likely to be mediated by the potentiation of PLCγ activation by phosphatidylinositol trisphosphate, either through Tec-family tyrosine kinases (26Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Google Scholar), Vav1 (27Manetz T.S. Gonzalez-Espinosa C. Arudchandran R. Xirasagar S. Tybulewicz V. Rivera J. Mol. Cell. Biol. 2001; 21: 3763-3774Google Scholar), or via direct effects on PLCγ (28Barker S.A. Lujan D. Wilson B.S. J. Leukocyte Biol. 1999; 65: 321-329Google Scholar, 29Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Google Scholar). A direct effect of phosphatidylinositol trisphosphate on calcium uptake has also been demonstrated (30Ching T.T. Hsu A.L. Johnson A.J. Chen C.S. J. Biol. Chem. 2001; 276: 14814-14820Google Scholar). However, an additional calcium-independent role of PI 3-kinases has been suggested by the fact that thapsigargin or calcium ionophore-mediated degranulation is still sensitive to PI 3-kinase inhibitors (15Kitani S. Teshima R. Morita Y. Ito K. Matsuda Y. Nonomura Y. Biochem. Biophys. Res. Commun. 1992; 183: 48-54Google Scholar,31Huber M. Hughes M.R. Krystal G. J. Immunol. 2000; 165: 124-133Google Scholar, 32Hirasawa N. Sato Y. Yomogida S. Mue S. Ohuchi K. Cell. Signal. 1997; 9: 305-310Google Scholar, 33Marquardt D.L. Alongi J.L. Walker L.L. J. Immunol. 1996; 156: 1942-1945Google Scholar). Our data show that p85/p110α is required for degranulation despite the fact that it is not required for antigen-stimulated calcium flux in RBL-2H3 cells (17Smith A.J. Surviladze Z. Gaudet E.A. Backer J.M. Mitchell C.A. Wilson B.S. J. Biol. Chem. 2001; 276: 17213-17220Google Scholar). This suggests that p85/p110α is a candidate for the wortmannin-sensitive, calcium-independent factor in antigen-stimulated degranulation. Our experiments also reveal a role for the p85/p110ॆ PI 3-kinase during adenosine-stimulated degranulation in cells treated with sub-optimal doses of antigen. The specific requirement for p85/p110ॆ in signaling by this Gαi-coupled receptor is consistent with reports that p110ॆ is activated by ॆγ subunits from trimeric G-proteins (34Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Google Scholar, 35Murga C. Fukuhara S. Gutkind J.S. J. Biol. Chem. 2000; 275: 12069-12073Google Scholar) and is required for lysophosphatidic acid-mediated signaling (36Roche S. Downward J. Raynal P. Courtneidge S.A. Mol. Cell. Biol. 1998; 18: 7119-7129Google Scholar). In addition to p110ॆ, p110γ is also required for adenosine secretion by mast cells and adenosine-mediated autocrine stimulation of degranulation in antigen-stimulated mast cells (18Laffargue M. Calvez R. Finan P. Trifilieff A. Barbier M. Altruda F. Hirsch E. Wymann M.P. Immunity. 2002; 16: 441-451Google Scholar). The coordination of these two Gॆγ-regulated PI 3-kinases during degranulation, and their potentially different downstream effectors, will be a subject of interest for future studies. We also studied degranulation in a heterologous system, the RBL-2H3-M1 cell line (11Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Google Scholar), which expresses the Gαq-coupled M1 muscarinic receptor. Previous studies have shown that carbachol-stimulated degranulation in these cells is different in several respects from FceRI-stimulated degranulation, for example with regard to the role of Src-family tyrosine kinases (37Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Google Scholar). Similarly, we find that inhibitors of PKCα/ॆ block degranulation in antigen-stimulated RBL-2H3 cells but not carbachol-stimulated RBL-2H3-M1 cells. Even given these differences, the requirement for hVPS34 during carbachol-stimulated degranulation of RBL-2H3-M1 cells is a surprising finding. The best documented roles for hVPS34 in mammalian cells are in the regulation of traffic through the endocytic or phagocytic systems (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar, 38Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.-C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell Biol. 1999; 1: 249-252Google Scholar, 39Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 1: 376-382Google Scholar, 40Murray J.T. Panaretou C. Stenmark H. Miaczynska M. Backer J.M. Traffic. 2002; 3: 416-427Google Scholar, 41Futter C.E. Collinson L.M. Backer J.M. Hopkins C.R. J. Cell Biol. 2001; 155: 1251-1264Google Scholar, 42Vieira O.V. Botelho R.J. Rameh L. Brachmann S.M. Matsuo T. Davidson H.W. Schreiber A. Backer J.M. Cantley L.C. Grinstein S. J. Cell Biol. 2001; 155: 19-25Google Scholar, 43Fratti R.A. Backer J.M. Gruenberg J. Corvera S. Deretic V. J. Cell Biol. 2001; 154: 631-644Google Scholar), and during the sorting of newly synthesized membrane proteins in polarized cells (44Tuma P.L. Nyasae L.K. Backer J.M. Hubbard A.L. J. Cell Biol. 2001; 154: 1197-1208Google Scholar). These reports are consistent with the well characterized role of VPS34p in vesicular trafficking in yeast (45Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Google Scholar). However, a role for hVPS34 during regulated secretion has not been demonstrated. It seems unlikely that hVPS34 is involved in degranulation at the level of granule fusion, since it would then also be required for degranulation in response to FcεRI receptor activation. Instead, our data suggest a novel role for hVPS34 in Gαq-coupled receptor signaling. How might hVPS34 modulate signaling from the M1 acetylcholine receptor? hVPS34 signals via the production of PI(3)P and the recruitment and/or activation of proteins containing FYVE or PX domains, which specifically bind to PI(3)P (46Stenmark H. Aasland R. J. Cell Sci. 1999; 112: 4175-4183Google Scholar, 47Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Google Scholar, 48Clague M.J. Urbe S. J. Cell Sci. 2001; 114: 3075-3081Google Scholar, 49Ellson C.D. Andrews S. Stephens L.R. Hawkins P.T. J. Cell Sci. 2002; 115: 1099-1105Google Scholar). In both mammalian cells and yeast, hVPS34 is targeting to membranes along with an associated protein kinase (VPS15/p150) (40Murray J.T. Panaretou C. Stenmark H. Miaczynska M. Backer J.M. Traffic. 2002; 3: 416-427Google Scholar, 45Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Google Scholar, 50Panaretou C. Domin J. Cockcroft S. Waterfield M.D. J. Biol. Chem. 1997; 272: 2477-2485Google Scholar); in mammalian cells the Rab5 GTPase also regulates hVPS34 targeting to endosomes (38Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.-C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell Biol. 1999; 1: 249-252Google Scholar, 40Murray J.T. Panaretou C. Stenmark H. Miaczynska M. Backer J.M. Traffic. 2002; 3: 416-427Google Scholar). Disruption of hVPS34 alters the post-endocytic trafficking of cell surface receptors (19Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Google Scholar, 41Futter C.E. Collinson L.M. Backer J.M. Hopkins C.R. J. Cell Biol. 2001; 155: 1251-1264Google Scholar) and therefore could affect the post-endocytic sorting of carbachol-stimulated M1 receptors. This could alter the signaling properties of the receptor, as has been suggested in the ॆ-adrenergic receptor system (51Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Google Scholar). Alternatively, a recently described RGS protein, RGS-PX1, is a Gαs-specific GAP that contains a PX domain (52Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Google Scholar); such a protein could provide a mechanism for the regulation of Gαs-coupled receptors by hVPS34. It is possible that PX- or FYVE-domain-containing RGS proteins might exist for Gαq-coupled receptors as well. Finally, it is possible that hVPS34 is a direct target of activated M1 receptors. However, we have so far been unable to detect changes in hVPS34 activity in immunoprecipitates from carbachol-stimulated RBL-2H3 cells (data not shown). In summary, we have used a single cell assay for mast cell degranulation to study the role of different PI 3-kinases in this process. We find a general role for Class IA PI 3-kinases in response to antigen-stimulated degranulation and a specific requirement for p85/p110ॆ during adenosine receptor-FcεRI receptor synergy. We also find an unexpected requirement for hVPS34 in carbachol-stimulated degranulation. These latter data suggest an unappreciated role for hVPS34 in signaling by Gαq-coupled receptors. It will be important to determine whether hVPS34 also plays a role in signaling by other G-protein-coupled receptors. We thank Dr. Bart Vanhaesebroeck, Ludwig Institute for Cancer Research, for anti-p110δ antibodies.
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