Receptor-Signaling Protein Interactions in Real Time

描述(申请人提供)：干扰素-伽马(干扰素-伽马)在免疫系统的调节中起关键作用，几乎对所有组织都有效。它的作用是由它的受体介导的，这是本应用的主题。我们以前发现了受体活性所需的两条链(干扰素-γ受体1和干扰素-γ受体2)。目前的建议集中在有无配体存在的情况下，活细胞中干扰素-γ受体复合体的链结构。针对这些目标的具体目标是：确定是否所有的干扰素-γ受体链都预先结合；JAK激酶在预组装中的作用；确定预组装受体链上的位点；STAT1在细胞内结构域的移动中的作用；确定配体结合后受体结构变化的动力学；分析下游信号成分与受体复合体的动力学和相互作用；确定作为干扰素-γ和干扰素-α受体复合体之间相互作用位点的受体。受体链的构型将通过活细胞中的荧光共振能量转移来研究。由于干扰素-γ在细胞和免疫功能中起着关键作用，了解其作用的所有这些方面将导致更好的策略来治疗各种疾病，如癌症、传染病和自身免疫性疾病。这项技术的进一步应用将导致高通量筛选和实时识别活细胞中蛋白质-蛋白质相互作用的新的快速方法。

DESCRIPTION (provided by applicant): Interferon gamma (IFN-gamma) plays a crucial role in regulation of the immune system and is active on virtually all tissues. Its actions are mediated by its receptor that is the subject of this application. We previously identified two chains (IFN-gammaR1 and IFN-gammaR2) that are required for receptor activity. The current proposal focuses on the structure of the chains of the IFN-gamma receptor complex in live cells in the presence and absence of ligand. The specific aims directed to these goals are: to determine if all the IFN-gamma, receptor chains preassociated; the role of the Jak kinases in preassembly; determination of the sites on the receptor chains for preassembly; role of Stat1 in movement of the intracellular domains; determination of the kinetics of change in receptor structure after ligand binding; analysis of the kinetics and interactions of the downstream signaling components with the receptor complex; determine the sites of the receptor that serve as sites for cross-talk between the IFN-gamma and IFN-alpha receptor complexes. The configuration of the receptor chains will be studied by fluorescence resonance energy transfer in live cells. Because IFN-gamma plays a critical role in cellular and immune function, understanding all these aspects of its action will lead to better strategies to attack a variety of diseases such as cancers, and infectious and autoimmune diseases. Further application of this technology should lead to new rapid methods for high throughput screening and for identifying protein-protein interactions in live cells in real-time.