Structure-function studies of visual arrestin

描述(申请人提供)：细胞对 持续性刺激，通常被称为脱敏，是一种广泛存在的生物学现象 现象。视觉放大级联(和其他G 蛋白质偶联受体)通过两步机制衰减： 视紫红质激酶对光激活视紫红质(Rh*)的磷酸化作用 其次是arrestin与光活化的磷酸化的紧密结合 视紫红质(P-Rh*)。Arrestin结合在信号关闭中的关键作用是 久负盛名。然而，支配分子机制的许多方面 不同类型光感受器细胞中Arrestin-受体的相互作用 有待澄清。 这项提议的目的是阐明分子机制。 负责杆状Arrestin与P-Rh*的优先结合 芦荟素向活性高亲和力转化的结构基础 视紫红质结合状态。Arrestin二聚化在其表达中的作用 光感受器的功能也将在体外和体内进行研究 使用具有增强和降低的突变倾向的 自我关联。视杆和视锥表达不同的arrestin蛋白， 视紫红质和视锥视觉色素(碘)的猝灭信号， 分别进行了分析。锥体arrestin活化的分子机制将是 与研究得更好的棒状芳香胺相比。杆件和锥件 阻滞剂是导致它们对视紫红质和碘紫质偏爱的原因， 将分别确定，以及它们在两者过渡中的作用 Arrestin蛋白将进入高亲和力受体结合状态 已澄清。已构建和新的具有结构性活性的“arrestin突变体” 与P-Rh*和Rh*都具有高亲和力的结合将被用来研究 棒中信号关闭和恢复的动力学。几种先天视力 视杆细胞中的视紫红质信号过多与疾病有关。 具有增强的关闭能力的结构性活性arrestin突变体 这种信号似乎是基因治疗这些疾病的合乎逻辑的工具。 这些突变体的治疗潜力将在这些模型中进行测试。 疾病，特别是表达视紫红质而缺乏视紫红质的小鼠 在视紫红质激酶基因敲除的小鼠中，发现 Arestin的代偿性变化是否能使他们的反应正常化 动力学和防止光依赖的视网膜退化。

DESCRIPTION (provided by applicant): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Visual amplification cascade (and signaling by other G protein-coupled receptors) is attenuated by a two-step mechanism: phosphorylation of light-activated rhodopsin (Rh*) by rhodopsin kinase, followed by tight binding of arrestin to light-activated phosphorylated rhodopsin (P-Rh*). The crucial role of arrestin binding in signal shut-off is well established. However, many aspects of the molecular mechanisms that govern arrestin-receptor interaction in different types of photoreceptor cells remain to be elucidated. The objectives of this proposal are to elucidate the molecular mechanism responsible for preferential binding of rod arrestin to P-Rh* and the structural basis of arrestin transition into its active high-affinity rhodopsin-binding state. The role of arrestin dimerization in its expression and function in photoreceptors will also be studied in vitro and in vivo with the use of mutants with an enhanced and reduced propensity for self-association. Rods and cones express different arrestin proteins that quench signaling by rhodopsin and cone visual pigments (iodopsins), respectively. The molecular mechanism of cone arrestin activation will be compared to that of a better studied rod arrestin. The elements of rod and cone arrestins responsible for their preference for rhodopsin and iodopsins, respectively, will be identified, and their role in the transition of both arrestin proteins into a high-affinity receptor-binding state will be elucidated. Already constructed and new constitutively active" arrestin mutants that bind with high affinity to both P-Rh* and Rh* will be used to study the kinetics of signal shut-off and recovery in rods. Several congenital vision disorders are associated with excessive rhodopsin signaling in rods. Constitutively active arrestin mutants with an enhanced ability to shut-off this signaling appear to be logical tools for gene therapy of these disorders. The therapeutic potential of these mutants will be tested in models of these disorders, in particular in mice expressing rhodopsin that lacks rhodopsin kinase phosphorylation sites and in rhodopsin kinase knock-out mice, to find out whether the compensatory change in arrestin can normalize their response kinetics and prevent light-dependent retinal degeneration.