Lyn Kinase Regulation of FceRI-induced Mediator Release

描述(申请人提供)：过敏反应的症状是 部分由过敏原诱导的预制以及 携带受体的嗜碱性粒细胞和肥大细胞合成的新介质 为了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和伽马链。Src家族酪氨酸激酶，Lyn，是 负责受体的磷酸化和磷酸化 以及众多下游信号分子的激活。我们最近做了 通过免疫复合酶检测和化学交联法显示 一小部分细胞Lyn与非磷酸化的IgE相关 肥大/嗜碱粒细胞系中的受体。各种方法，包括 多肽结合研究、酵母双杂交相互作用及转染法 完整的细胞表明Lyn的独特结构域与CTerus相关 受体的β链[β(Fc)]。Lyn-FcERI的颠覆 与转染型Lyn独有结构域的相互作用完全阻止受体 受体聚集时酪氨酸的磷酸化。我们已经测试了它的能力 转基因LYN独特结构域在防止啮齿类动物组胺释放(HR)中的作用 肥大/嗜碱粒细胞系(RBL)。检测到40%的心率抑制 转染体稳定表达比内源性多5倍的独特结构域 将LYN与载体转染的RBL细胞进行比较。我们建议将这些措施延长 通过检测白三烯C4和肿瘤坏死因子-α在我们的RBL中的产生进一步研究 转染者。此外，我们建议进一步定义Lyn-beta(FC) Lyn独特结构域的定点突变相互作用区 然后在RBL细胞中进行转染。完整的LYN之间的直接相互作用 独特的结构域和β(Fc)将通过表面等离子激元共振进行评估。这个 相互作用的特异性将通过LYN的转染来评估 与其他含Src家族激酶的细胞的唯一结构域及其相互作用 具有适当的受体亚单位(T细胞和T细胞抗原受体Zeta， B细胞和B细胞抗原受体IgA，嗜酸性粒细胞和IL-5受体共同 用免疫共沉淀法和Western blotting进行鉴定。交付一份 来自Lyn独特结构域的多肽将在人类身上进行评估 使用脂肪酸修饰、穿透性多肽或 森姆利基森林病毒。理解相互作用的分子细节 LYN的独特结构域和FcERIβ(Fc)可能允许药物的开发 这将抑制过敏反应，而不是缓解他们的症状。

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.