Src Family Kinase Regulation in Allergy

过敏原诱导高亲和力IgE受体聚集后炎症介质的分泌 肥大细胞上的FceRI（FceRI）是哮喘和其他过敏性疾病的发病机制的关键组成部分。 Src家族酪氨酸激酶林恩和Fyn均正向和负向调节FceRI信号传导， 肥大细胞的分泌。我们推测，林恩和费恩在调节神经元细胞凋亡方面的功能差异， 肥大细胞分泌是由于它们独特的Src同源（SH）结构域差异地 与FceRI信号传导的正或负调节因子相关。此外，我们假设， 将林恩区室化为膜筏有助于其参与负调节激活 Fyn kinase激酶具体目的是：1）在骨髓中表达显性阴性（DN）林恩突变体 衍生的肥大细胞（BMMC），并测量抗原诱导的介质分泌，信号转导的变化， 林恩和FceRI之间的关联。为了确定FceRI β上林恩结合的位点， 非聚集受体将林恩A或林恩B重新引入林恩-/- BMMC中，以研究特异性 每个同种型对FceRI信号传导、趋化性和分泌的贡献。2)为了表达DN Fyn突变体， BMMC和测量抗原诱导的介质分泌、信号转导变化和Fyn之间的关联 和FceRI。确定FceRI β上Fyn与未聚集受体的结合位点。第三章 为了直接测量林恩与活细胞中FceRI β和γ亚基缔合的能力，使用 荧光共振能量转移比较筏式定位和筏式排除的活性和功能 林恩激酶对FceRI信号传导和分泌的影响。 与公共卫生的相关性：西方国家过敏性疾病的发病率正在增加， 现在全世界的治疗费用总计数十亿美元。了解相互作用的分子细节 Src家族激酶与FceRI的结合可能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.