Src Family Kinase Regulation in Allergy

变应原诱导高亲和力IgE受体聚集后炎性介质的分泌 肥大细胞(FceRI)是哮喘和其他过敏性疾病发病机制的关键组成部分。 Src家族酪氨酸激酶Lyn和Fyn对FceRI信号有正向和负向调节作用。 肥大细胞的分泌物。我们假设LYN和FYN在调节方面的功能差异 肥大细胞分泌源于其独特的和Src同源(SH)结构域的能力 与FceRI信号的正向或负向调节因子有关。此外，我们假设 LYN区隔成膜筏便于其参与负性调节激活 Fyn Kinase的。其具体目的是：1)在骨髓中表达显性阴性(DN)Lyn突变体 来源的肥大细胞(BMMC)并测量抗原诱导的介质分泌、信号变化和 LYN和FceRI之间的联系。要确定LYN在FceRI测试版上的结合位置，请使用 未聚集的受体。将LYN A或LYN B重新引入LYN-/-BMMC以研究特定的 每种异构体对FceRI信号、趋化和分泌的贡献。2)表达DNFYN突变体 BMMC与测量抗原诱导的介质分泌、信号变化及Fyn之间的关联 和FceRI。确定FceRIβ上Fyn与非聚集受体的结合位置。3) 为了直接测量Lyn与活细胞中FceRIβ和Gamma亚基的结合能力，使用 荧光共振能量转移。比较局部RAFT和RAFT-INCLUDE的活性和功能 Lyn激酶对FceRI信号和分泌的影响。 与公共卫生的相关性：在西化国家，过敏性疾病的发病率正在增加， 目前，全球治疗费用总计达数十亿美元。理解相互作用的分子细节 与FceRI一起使用Src家族激酶可能会允许开发限制过敏反应的药物 而不是减轻症状。

Secretion of inflammatory mediators after allergen-induced aggregation of the high affinity IgE receptor (FceRI) on mast cells is a critical component of the pathogenesis of asthma and other allergic disorders. The Src family tyrosine kinases, Lyn and Fyn, both positively and negatively regulate FceRI signaling and secretion in mast cells. We hypothesize that the functional differences between Lyn and Fyn in regulating mast cell secretion arise from the ability of their unique and Src homology (SH) domains to differentially associate with positive or negative regulators of FceRI signaling. Furthermore, we hypothesize that the compartmentalization of Lyn into membrane rafts facilitates its participation in negatively regulating activation of Fyn kinase. The specific aims are 1) to express dominant negative (DN) Lyn mutants in bone marrow derived mast cells (BMMC) and measure antigen-induced mediator secretion, signaling changes and association between Lyn and the FceRI. To determine the site of Lyn association on the FceRI beta with unaggregated receptors. To reintroduce either Lyn A or Lyn B into Lyn -/- BMMC to investigate the specific contribution of each isoform to FceRI signaling, chemotaxis and secretion. 2) To express DN Fyn mutants in BMMC and measure antigen-induced mediator secretion, signaling changes and association between Fyn and the FceRI. To determine the site of Fyn association on the FceRI beta with unaggregated receptors. 3) To directly measure the ability of Lyn to associate with FceRI beta and gamma subunits in living cells using fluorescence resonance energy transfer. To compare the activity and functions of raft-localized and raftexcluded Lyn kinase on FceRI signaling and secretion. Relevance to public health: The incidence of allergic disease in Westernized countries is increasing and treatment costs worldwide now total billion of dollars. Understanding the molecular details of the interaction of Src family kinases with FceRI may allow the development of drugs that will limit allergic reactions rather than mitigating symptoms.