Lyn Kinase Regulation of FceRI-induced Mediator Release

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DESCRIPTION (provided by applicant): The symptoms of an allergic reaction are generated in part by the allergen-induced release of preformed as well as newly synthesized mediators from basophils and mast cells bearing receptors for IgE. Initiation of signal transduction through the high affinity receptor for IgE (FcERI) requires phosphorylation of distinct tyrosine residues in the receptor's beta and gamma chains. The Src-family tyrosine kinase, Lyn, is responsible for the receptor phosphorylation as well as the phosphorylation and activation of numerous downstream signaling molecules. We have recently shown through immune complex kinase assays and chemical crosslinking that a small fraction of cellular Lyn is associated with unphosphorylated IgE receptors in mast/basophil cell lines. A variety of approaches, including peptide binding studies, yeast two-hybrid interaction and transfection of intact cells indicate that the unique domain of Lyn is associated with the Cterminus of the receptor's beta chain [beta(Fc)]. Disruption of the Lyn-FcERI interaction with transfected Lyn unique domain completely prevented receptor tyrosine phosphorylation upon receptor aggregation. We have tested the ability of transfected Lyn unique domain to prevent histamine release (HR) in a rodent mast/basophil cell line (RBL). Forty percent inhibition of HR was detected in transfectants stably expressing five times more unique domain than endogenous Lyn compared to vector-transfected RBL cells. We propose to extend these studies further by examining leukotriene C4 and TNF-a production in our RBL transfectants. In addition, we propose to further define the Lyn-beta(Fc) interacting region by site-directed mutagenesis of the Lyn unique domain followed by transfection in RBL cells. Direct interaction between intact Lyn unique domain and beta(Fc) will be assessed by surface plasmon resonance. The specificity of the interaction will be evaluated by transfection of the Lyn unique domain into other Src-family kinase containing cells and association with an appropriate receptor subunit (T cell and T cell antigen receptor zeta, B cell and B cell antigen receptor Iga, eosinophil and IL-5 receptor common beta) evaluated by co-immunoprecipitation and Western blotting. Delivery of a peptide derived from the Lyn unique domain will be evaluated in human basophils using either fatty acid modification, a Penetratin peptide or Semliki forest virus. Understanding the molecular details of the interaction of the Lyn unique domain and FcERI beta(Fc) may allow the development of drugs that will inhibit allergic reactions rather than mitigating their symptoms.