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FcepsilonRI的信号是由受体交联启动的，并通过两种蛋白酪氨酸激酶的顺序激活而传播：LYN，它磷酸化FcepsilonRIβ和伽马亚基中基于免疫受体酪氨酸的激活基序(ITAM)中的酪氨酸；以及Syk，它被招募到伽马亚单位磷酸化，进而激活导致过敏、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)的珍妮特·奥利弗(Janet Oliver)博士和墨西哥国立自治大学(UNAM)的恩里克·奥尔特加(Enrique Ortega)博士一直在FIRCA支持的合作研究中，对RBL-2H3肥大细胞中这种激酶级联的调控进行研究。他们的工作使用了独特的抗FcepsilonRI单抗，与多价抗原相比，这些单抗具有信号传递能力(MAbJ17)或受损(MAbH10)。他们最近的结果将H10诱导的二聚体的微弱信号活性与其未能完成Lyn介导的FcepsilonRIβ亚基磷酸化系列联系在一起，这显然是Lyn解离、Syk募集和信号传播所必需的。更新申请的目的1测试假设，LYN与部分磷酸化的FcepsilonRI亚基相互作用，必须从完全磷酸化的受体上移位，才能允许Syk募集和信号传播。拟议的实验包括单抗处理的RBL-2H3细胞中磷酸β的磷酸氨基酸分析，以及基于生化和荧光的LYN S与合成的磷酸β亚型结合的测量。更新申请的目的2验证了这一假说，即交联型FcepsilonRI在与Lyn丰富的、洗涤剂不溶的膜微区短暂结合时遇到Lyn，必须完成释放Lyn的磷酸化系列，才能重新分布到主体膜并与Syk和下游信号分子相互作用。该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态。这一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递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.