Lyn Kinase Regulation of FceRI-induced Mediator Release

描述（由申请人提供）：过敏反应的症状是 部分是由变应原诱导的预制的释放产生的， 嗜碱性粒细胞和肥大细胞携带受体新合成的介质 对于IgE。通过高亲和力受体启动信号转导 对于IgE（FcERI），需要磷酸化不同的酪氨酸残基， 受体的β和γ链。Src家族酪氨酸激酶，林恩， 负责受体的磷酸化， 以及许多下游信号分子的激活。我们最近 通过免疫复合物激酶测定和化学交联显示， 一小部分细胞林恩与非磷酸化IgE相关 肥大细胞/嗜碱性细胞系中的受体。各种方法，包括 肽结合研究，酵母双杂交相互作用和转染 完整的细胞表明林恩的独特结构域与C末端相关 受体β链[beta（Fc）]的一部分。Lyn-FcERI的破坏 与转染林恩独特结构域的相互作用完全阻止了受体 酪氨酸磷酸化后受体聚集。我们测试了 转染的林恩独特结构域，以防止啮齿动物中组胺释放（HR） 肥大/嗜碱性细胞系（RBL）。40%的HR抑制被检测到， 稳定表达比内源性多五倍的独特结构域的转染子 林恩相比，载体转染的RBL细胞。我们建议延长这些 通过检测我们的RBL中白三烯C4和TNF-α的产生， 转染子此外，我们建议进一步定义Lyn-beta（Fc） 通过林恩独特结构域的定点诱变的相互作用区 随后转染RBL细胞。完整的林恩之间的直接相互作用 独特结构域和β（Fc）将通过表面等离子体共振进行评估。的 相互作用的特异性将通过转染林恩 独特的结构域进入其他含Src家族激酶的细胞， 与适当的受体亚单位（T细胞和T细胞抗原受体ζ， B细胞和B细胞抗原受体伊加、嗜酸性粒细胞和IL-5受体共同 β）通过免疫共沉淀和蛋白质印迹法评价。递送 将在人体中评价源自林恩独特结构域的肽 使用脂肪酸修饰、Penetratin肽或 塞姆利基森林病毒。了解相互作用的分子细节 林恩独特结构域和FcERI β（Fc）的结合可能允许开发药物 抑制过敏反应而不是减轻症状。

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.