Structure-function studies of visual arrestin

项目摘要 我们建议阐明arrestin-1与视紫红质相互作用的分子机制， 显著有助于感光细胞的精细调节和精确定时功能，使用 结合新的尖端生物物理方法和体内实验。我们将比较 使用在所选点之间的长距离测量的arrestin-1与视紫红质的复合物 arrestin-1和视紫红质的脉冲EPR技术DEER。我们将探讨抑制蛋白结合的构象 视紫红质的构象，并将其与游离光激活视紫红质和与 转导以确定视紫红质的特定构象状态（模型GPCR）是否使其倾向于 与G蛋白和抑制蛋白相互作用。这在本领域中被假设为偏置GPCR信号传导的基础。 我们将确定arrestin-1自缔合在体内光感受器中的生物学作用，目前， 未知最后，我们将测试功能获得性视紫红质基因治疗的新补偿方法 使用能够关闭视紫红质信号传导的工程化增强型arrestin-1突变体的突变 独立于其磷酸化。拟议的研究将大大提高我们对 光感受器生理学的研究进展，并推进我们在功能获得性视紫红质基因治疗方面的进展 突变。由于GPCR调控机制的高度保守性，抑制蛋白-1的机制研究 结合到视紫红质将有更广泛的影响，提高我们的理解的分子基础， 其他arrestin亚型的功能。拟议中的研究将为细胞信号传导的许多方面提供新的线索。我们 战略目标是充分了解蛋白质-蛋白质相互作用，控制细胞信号传导， 构建具有所需功能特征的突变体用于研究和治疗目的。

PROJECT SUMMARY We propose to elucidate the molecular mechanisms of arrestin-1 interactions with rhodopsin, which significantly contribute to exquisitely regulated and precisely timed function of photoreceptor cells, using the combination of novel cutting-edge biophysical methods and in vivo experiments. We will compare the shape of the complex of arrestin-1 with rhodopsin using long-range distance measurements between selected points in arrestin-1 and rhodopsin with pulse EPR technique DEER. We will explore the conformation of arrestin-bound rhodopsin and compare it to the conformation of free light-activated rhodopsin and rhodopsin in complex with transducing to determine whether particular conformation states of rhodopsin (a model GPCR) predispose it to interact with G protein and arrestin. This is hypothesized in the field to be the basis of biased GPCR signaling. We will determine the biological role of arrestin-1 self-association in photoreceptors in vivo, which is currently unknown. Finally, we will test novel compensational approach to gene therapy of gain-of-function rhodopsin mutations using engineered enhanced arrestin-1 mutants capable of shutting off rhodopsin signaling independently of its phosphorylation. Proposed studies will significantly improve our understanding of photoreceptor physiology and advance our progress towards gene therapy of gain-of-function rhodopsin mutations. Due to high conservation of mechanics of GPCR regulation, mechanistic studies of arrestin-1 binding to rhodopsin will have broader implications, improving our understanding of the molecular basis of the function of other arrestin subtypes. Proposed studies will shed new light on many aspects of cell signaling. Our strategic goal is to gain sufficient understanding of protein-protein interactions that govern cell signaling to construct mutants with desired functional characteristics for research and therapeutic purposes.