STUCTURE-FUNCTION RELATIONSHIPS OF IMMUNORECEPTORS

在免疫应答中介导细胞活化的多链免疫识别受体（MIRR）包括抗原的T细胞受体、B细胞受体和Fc受体。将使用免疫球蛋白E（IgE）的高亲和力Fc受体（FcepsilonRI）作为范例，研究质膜MIRR功能的结构基础，免疫球蛋白E（IgE）在过敏性免疫应答中起着核心作用。拟议的研究将集中在质膜异质性，动力学和结构，因为这些都参与了IgE-FcepsilonRI信号转导，特别是液体有序的，洗涤剂抗性区域在FcepsilonRI和林恩酪氨酸激酶之间的初始偶联中发挥的作用。具体目标1将使用几种不同的嵌合受体，结合位点特异性诱变和胆固醇光亲和标记研究，检查FcepsilonRI结构的特征，这些特征对于其与耐洗涤剂膜的相互作用至关重要。具体目标2将应用先进的生物物理方法，包括定量荧光显微镜、电子自旋共振和质谱法，以表征全细胞和亚细胞制剂质膜中的这些胆固醇依赖性、去污剂抗性区域;将研究细胞活化导致的结构变化。例如，使用绿色荧光蛋白缀合的类似物在完整细胞上监测锚定到质膜的内小叶的林恩和抗原聚集的Fc ε RI之间的真实的相互作用，以检测荧光共振能量转移的邻近性变化和荧光相关光谱和成像的迁移率变化。还将研究F-肌动蛋白与质膜相互作用的结构基础及其对FcepsilonRI信号传导的调节。这些研究将检验一般假设，即质膜的结构组织对于免疫应答中受体介导的信号传导是重要的。

The multi-chain immune recognition receptors (MIRR) which mediate cell activation in the immune response include T cell receptors for antigen, B cell receptors, and Fc receptors. The structural basis for MIRR function at the plasma membrane will be investigated, using as a paradigm the high affinity Fc receptor (FcepsilonRI) for immunoglobulin E (IgE) which plays a central role in the allergic immune response. Proposed studies will focus on plasma membrane heterogeneity, dynamics and structure as these are involved in IgE-FcepsilonRI signaling, and in particular on the role liquid-ordered, detergent-resistant regions play in the initial coupling between FcepsilonRI and Lyn tyrosine kinase. Specific aim 1 will examine features of FcepsilonRI structure that are critical for its interaction with detergent resistant membranes, using several different chimeric receptors, together with site-specific mutagenesis and cholesterol photoaffinity labeling studies. Specific aim 2 will apply advanced biophysical methods including quantitative fluorescence microscopy, electron spin resonance and mass spectrometry to characterize these cholesterol-dependent, detergent-resistant regions in plasma membranes of whole cells and sub-cellular preparations; structural changes that occur as a result of cell activation will be investigated. For example, real time interactions between Lyn anchored to the inner leaflet of the plasma membrane and antigen-aggregated FcepsilonRI will be monitored on intact cells using green fluorescent protein-conjugated analogues to detect proximity changes with fluorescence resonance energy transfer and mobility changes with fluorescence correlation spectroscopy and imaging. The structural basis for the interaction of F- actin with the plasma membrane and its regulation of FcepsilonRI signaling will also be investigated. These studies will test te general hypothesis that structural organization at the plasma membrane is important for receptor-mediated signaling in the immune response.