IGE RECEPTOR DIMERS--STRUCTURAL AND SIGNALING PROPERTIES

通过嗜碱性粒细胞和肥大细胞的高亲和力IgE受体Fc ε RI的信号传导由受体交联引发，并通过两种蛋白酪氨酸激酶的顺序活化而传播：林恩，其使Fc ε RI β和γ亚基中基于免疫受体酪氨酸的活化基序（ITAM）内的酪氨酸磷酸化;和Syk，其被募集到γ亚基磷酸化ITAM，并进而激活导致过敏、哮喘和过敏反应的信号传导途径。 自1994年8月以来，新墨西哥州大学（UNM）的Janet奥利弗博士和墨西哥国立自治大学（UNAM）的Enrique Ortega博士进行了FIRCA支持的关于RBL-2 H3肥大细胞中该激酶级联调节的合作研究。 他们的工作使用了独特的抗FcepsilonRI单克隆抗体，与多价抗原相比，这些抗体具有信号传导能力（mAb J17）或受损（mAb H10）。 他们最近的结果将H10诱导的二聚体的弱信号传导活性与它们未能完成林恩解离、Syk募集和信号传播明显所需的Lyn-mediated FceppsilonRI β亚基磷酸化系列联系起来。 更新申请的目的1测试了以下假设：林恩与部分磷酸化的Fc ε RI亚基相互作用，并且必须从完全磷酸化的受体中置换以允许Syk募集和信号传播。 拟定的实验包括mAb处理的RBL-2 H3细胞中磷酸-β的磷酸氨基酸分析以及林恩与合成磷酸-β同种型结合的生物化学和基于荧光的测量。 更新申请的目的2检验了以下假设：交联的FcepsilonRI在与富含Lyn的洗涤剂不溶性膜微结构域短暂结合期间遇到林恩，并且必须完成释放林恩的磷酸化系列，以便重新分布到主体膜中并与Syk和下游信号分子相互作用。 拟议的实验探索膜分离和荧光共振能量转移的受体和激酶的膜拓扑结构。这项持续研究的结果将为通过免疫细胞的抗原受体控制信号传导的途径提供新的见解。

Signaling through the high affinity IgE receptor, FcepsilonRI, of basophils and mast cells is initiated by receptor crosslinking and propagated by the sequential activation of two protein tyrosine kinases: Lyn, that phosphorylates tyrosines within immunoreceptor tyrosine-based activation motifs (ITAMs) in the FcepsilonRI beta and gamma subnuits; and Syk, that is recruited to the gamma subunit phospho-ITAMs and in turn activates signaling pathways leading to allergic, asthmatic and anaphylactic reactions. Since August, 1994, Dr. Janet Oliver from the University of New Mexico (UNM) and Dr. Enrique Ortega from the Universidad Nacional Autonoma de Mexico (UNAM) have conducted FIRCA-supported collaborative research on the regulation of this kinase cascade in RBL-2H3 mast cells. Their work uses unique anti-FcepsilonRI monoclonal antibodies that are signaling competent (mAb J17) or impaired (mAb H10) in comparison with multivalent antigen. Their recent results link the weak signaling activity of H10-induced dimers to their failure to complete a Lyn-mediated FcepsilonRI beta subunit phosphorylation series apparently required for Lyn dissociation, Syk recruitment and signal propagation. Aim 1 of the renewal application tests the hypothesis that Lyn interacts with partially phosphorylated FcepsilonRI subunits and must be displaced from fully phosphorylated receptors in order to permit Syk recruitment and signal propagation. The proposed experiments consist of phosphoamino acid analyses of phospho-beta from mAb-treated RBL- 2H3 cells and biochemical and fluorescence-based measurements of Lyn s binding to synthetic phospho-betaisoforms. Aim 2 of the renewal application tests the hypothesis that crosslinked FcepsilonRI encounter Lyn during their transient association with Lyn-enriched, detergent-insoluble membrane microdomains and must complete the phosphorylation series that releases Lyn in order to redistribute into bulk membrane and interact with Syk and downstream signaling molecules. The proposed experiments explore the membrane topography of receptors and kinases by membrane fractionation and fluorescence resonance energy transfer. Results of this continued research will provide new insights into the pathways controlling signaling through the antigen receptors of immune cells.