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T细胞抗原受体、B细胞受体和Fc受体。将以免疫球蛋白E(IgE)的高亲和力Fc受体(FcepsilonRI)为范例，研究质膜MirR功能的结构基础，免疫球蛋白E在过敏免疫反应中发挥核心作用。建议的研究将集中在质膜的异质性、动力学和结构上，因为这些都涉及到IgE-FcepsilonRI信号，特别是液体有序的洗涤剂抗性区域在FcepsilonRI和Lyn酪氨酸激酶之间的初始偶联中所起的作用。具体目标1将使用几种不同的嵌合受体，结合定点突变和胆固醇光亲和标记研究，检查FcepsilonRI结构的特征，这些特征对其与洗涤剂耐受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学，来表征全细胞和亚细胞制剂质膜中这些依赖胆固醇、抵抗洗涤剂的区域；将研究由于细胞激活而发生的结构变化。例如，将使用绿色荧光蛋白结合类似物在完整细胞上监测锚定在质膜内叶上的LYN与抗原聚集的FcepsilonRI之间的实时相互作用，以通过荧光相关光谱和成像来检测与荧光共振能量转移和迁移率变化的邻近变化。此外，还将探讨F-肌动蛋白与质膜相互作用的结构基础及其对FcepsilonRI信号的调控。这些研究将检验一个普遍的假设，即质膜上的结构组织对于免疫反应中受体介导的信号传递是重要的。

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.