STRUCTURE - FUNCTION RELATIONSHIPS OF THE IGE RECEPTOR

所提出的工作是三维分析的继续 免疫球蛋白E(IgE)受体复合体的结构及其相互作用 和其他细胞成分，以及它传递 穿过肥大细胞和嗜碱性粒细胞质膜的信号触发 过敏反应中的脱颗粒。距离的结构映射 在免疫球蛋白E、受体和膜表面的明确位置之间 将继续使用共振能量传递方法，并推出一种新的 这项技术的方法将使用荧光标记进行开发 针对IgE-受体复合体中的位点的单抗。 单克隆体对细胞脱颗粒的功能影响将是 经过测试，并与其结构位置相关联。生化研究 关于膜结合受体的研究将旨在延长之前 关于β和伽马亚基胞质暴露的信息 通过检测膜结合受体对限制性的敏感性 蛋白质分解消化。其他有关细胞结构的生化研究 免疫球蛋白受体复合体将包括光亲和标记a的尝试。 受体上潜在的蛋白激酶位点及其分离和Fc的努力 啮齿动物免疫球蛋白E与受体结合的片段。对这一概念的理解 IgE与受体相互作用的细节最终应该会有所帮助 在受体特异性抗过敏药物的开发中。 细胞上受体大规模聚集的最新观察 由小分子IgE寡聚体诱导的表面将通过 荧光光漂白回收法定量测量 和共振能量传递方法。这些方法应该允许 在以下条件下，聚团的动力学分析 与脱颗粒有关，使得两个粒子之间的时间关系 可以对流程进行评估。生化研究将针对 识别与群集交互的蜂窝组件 受体，从而可能参与跨膜信号。为 这些目的是化学交联和选择性细胞骨架 解聚方法将用于分离与受体相关的 洗涤剂溶解细胞中的成分。这些研究应该 为结构性和功能性事件链提供新的见解 细胞表面IgE受体复合体的抗原桥联与 脱颗粒信号。

The work proposed is a continuation of analyses of the three-dimensional structure of the immunoglobulin E(IgE)-receptor complex, its interactions with other cellular components, and the mechanism by which it transmits a signal across the plasma membrane of mast cells and basophils to trigger degranulation in allergic responses. Structural mapping of distances between well-defined sites in IgE, the receptor, and the membrane surface will be continued using resonance energy transfer methods, and a new approach to this technique will be developed using fluorescently-labeld monoclonal antibodies specific for sites in the IgE-receptor complex. Functional effects of the monoclonals on cellular degranulation will be tested and correlated to their structural locations. Biochemical studies on the membrane -bound receptor will be aimed at extending previous information about the cytoplasmic exposure of the Beta and Gamma subunits by examining the susceptibility of membrane-bound receptor to limited proteolytic digestion. Other biochemical studies on the structure of the IgE-receptor complex will include an attempt to photoaffinity label a potential protein kinase site on the receptor and efforts to isolate and Fc fragment of rodent IgE that binds to the receptor. An understanding of the details of the interaction of IgE with the receptor should ultimately aid in the development of receptor-specific anti-allergic pharmaceuticals. Recent observations of large-scale clustering of receptors on the cell surface that is induced by small oligomers of IgE will be extended by making quantitative measurements with fluorescence photobleaching recovery and resonance energy transfer methods. These approaches should permit analysis of the kinetics of the clustering under conditions that are relevant to degranulation such that the temporal relationship between two processes may be assessed. Biochemical studies will be directed toward the identification of cellular components that interact with clustered receptors and thereby may be involved in the transmembrane signal. For these purposes chemical cross-linking and selective cytoskeletal depolymerigation methods will be used to isolate receptor-associated components from detergent-solubilized cells. These studies should contribute new insights to structural and functional chain-of-events between antigen bridging of IgE-receptor complexes on the cell surface and the degranulation signal.