Molecular analysis of FceRI signaling and function

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

The high affinity receptor for IgE (FceRI) is central to allergic reactions. It is a tetrameric structure (a[3y2) when expressed on mast cells and basophils. The c_ chain is solely responsible for binding the ligand IgE while the [3 and 7 chains are involved in signaling. Aggregation of Fc_RI by IgE and the corresponding multivalent antigen induces the release of allergy mediators and proinflammatory cytokines. One of the critical events that connect Fc_RI aggregation with mediator and cytokine secretion is Ca z+ mobilization. Ca z¿ mobilization is comprised of Ca 2¿ release from intracellular stores, and Ca 2¿ entry from the extracellular milieu. The role played by phospholipase Cy in contributing to Ca 2¿ release from intracellular stores is welt documented. It produces the second messenger 1,4,5-triphosphate (IP3) that binds to IP 3 receptors, thereby inducing Ca 2+ release from intracellular stores. A few years ago we showed that, in addition to the well- known role played by phospholipase Cy, sphingosine 1-phosphate (S 1P) was critical in Ca 2+ release induced by FcERI. S1P is produced by the action of a sphingosine kinase activity on sphingosine. Two sphingosine kinases have been cloned but they have not been shown to be involved in the Ca 2+ release induced by Fc_RI aggregation. In this proposal our central hypothesis is that Fc_RI-dependent Ca 2+ release requires both PLCy and PI3KC21_ activation and that PI3KC21_ plays a major role in mast cell function. We present preliminary data showing that PI3KC213 possesses SK activity, is activated after FcERI aggregation, and contributes significantly to Ca z¿ release. In Aim 1 we will elucidate the mechanisms of FceRI-mediated activation of the PI3KC213 sphingosine kinase activity. In Aim 2 we will dissect the structural requirements underlying the sphingosine kinase function of PI3KC213.In Aim 3 we will elucidate the role of sphingosine kinase function of PI3KC213 in mast cell development and mast cell function in vitro and in vivo using mice with a mast cell specific PI3KC213 deficiency. All together, the proposed experiments will provide a comprehensive characterization of PI3KC213 and its sphingosine kinase activity, and define its role in mast cell physiology.