IGE RECEPTOR DIMERS--STRUCTURAL AND SIGNALING PROPERTIES

嗜碱性粒细胞和肥大细胞的高亲和IgE受体FcepsilonRI的信号通过受体交联启动，并通过两种蛋白酪氨酸激酶的顺序激活来传播：Lyn，其磷酸化FcepsilonRI β和γ亚基中免疫受体酪氨酸基激活基序（ITAMs）中的酪氨酸；和Syk，它被招募到γ亚基磷酸itams，反过来激活导致过敏、哮喘和过敏反应的信号通路。自1994年8月以来，新墨西哥大学（UNM）的Janet Oliver博士和墨西哥国立自治大学（UNAM）的Enrique Ortega博士就RBL-2H3肥大细胞中这种激酶级联的调控进行了firca支持的合作研究。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，这些单克隆抗体具有信号传导能力（mAb J17）或受损（mAb H10）。他们最近的研究结果将h10诱导的二聚体的弱信号活性与它们无法完成Lyn介导的FcepsilonRI β亚基磷酸化系列联系起来，这显然是Lyn解离、Syk募集和信号传播所必需的。更新应用的目的1验证了Lyn与部分磷酸化的FcepsilonRI亚基相互作用的假设，并且必须从完全磷酸化的受体中移位，以允许Syk募集和信号传播。实验包括从单克隆抗体处理的RBL- 2H3细胞中分析磷酸- β的磷酸氨基酸，以及生化和荧光检测Lyn s与合成磷酸- β异构体的结合。更新应用的目的2验证了这样的假设，即交联的FcepsilonRI在与富含Lyn的、不溶于洗涤剂的膜微域的短暂结合过程中遇到Lyn，并且必须完成释放Lyn的磷酸化系列，才能重新分配到大膜中，并与Syk和下游信号分子相互作用。本实验通过膜分离和荧光共振能量转移来探索受体和激酶的膜形貌。这项持续研究的结果将为通过免疫细胞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.