Understanding signaling through the heterotrimeric (alphabetagamma2) IgE receptor, FcepsilonRI, of basophils and mast cells is critical to developing new treatments for allergic inflammation. Work supported by grant GM-49814 identified key steps in a signaling cascade in which FcepsilonRI crosslinking induces the Lyn-mediated tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the FcepsilonRI beta and gamma subunits, providing phospho-tyrosine binding sites for a second tyrosine kinase, Syk, responsible for signal propagation. Recent studies suggest that Lyn is topographically restricted in the mast cell membrane, that Lyn dissociation from fully phosphorylated FcepsilonRI beta subunits is a rate-limited step (and potential therapeutic target) for Syk recruitment, and that Lyn plays negative as well as positive roles in FcepsilonRI- coupled signal transduction. In this renewal application, Specific Aim 1 uses a new electron microscopic method to test the hypothesis that Lyn associates with lipid microdomains rather than with FcepsilonRI in resting mast cells and to determine the topographical relationships of receptor, Lyn and Syk to each other and to clathrin-coated membrane during the signaling cycle.
Specific Aim 2 uses sequence-specific anti- phosphotyrosine antibodies to test the hypothesis that FcepsilonRI crosslinking induces an orderly sequence of FcepsilonRI beta ITAM phosphorylation; a panel of synthetic peptides to test the hypothesis that Lyn has greater binding activity towards partially than fully phosphorylated FcepsilonRI beta ITAMs; and transfection of wild-type and mutagenized beta subunits to determine if human FcepsilonRI signaling can be arrested by tyrosine substitutions that arrest the FcepsilonRI beta ITAM phosphorylation sequence.
Specific Aim 3 uses Lyn-/- mast cells to explore negative as well as positive roles for Lyn in FcepsilonRI-coupled signal transduction and to determine if negative signaling is mediated in part by the lipid phosphatase, SHIP. Results of these studies may reveal new therapeutic targets among the cascade of events that initiate, propagate and modulating through the FcepsilonRI and related receptors.
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