Structure-function studies of visual arrestin

描述（由申请人提供）：细胞对A的反应性降低， 持续性刺激，通常称为脱敏，是一种广泛的生物学反应， 现象视觉放大级联（和其他G 蛋白偶联受体）通过两步机制减弱： 光激活视紫红质（Rh*）被视紫红质激酶磷酸化， 随后抑制蛋白与光激活的磷酸化 视紫红质（P-Rh*）。抑制蛋白结合在信号关闭中的关键作用是 很好地建立了。然而，许多方面的分子机制， 不同类型的感光细胞中的抑制蛋白-受体相互作用仍然存在 有待阐明。 本提案的目标是阐明分子机制 负责杆抑制蛋白与P-Rh* 的优先结合， arrestin向其活性高亲和力转变结构基础 视紫红质结合态抑制蛋白二聚体在其表达中的作用 在光感受器中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倾向的突变体， 自我联想视杆细胞和视锥细胞表达不同的抑制蛋白， 通过视紫红质和视锥细胞色素（碘视蛋白）猝灭信号， 分别锥抑制蛋白激活的分子机制将是 与更好研究的杆抑制蛋白相比。杆锥元素 抑制蛋白负责它们对视紫红质和碘视蛋白的偏好， 将分别确定，他们在两者的过渡中的作用 将抑制蛋白转化为高亲和力受体结合状态， 阐明。已经构建的和新的组成型活性”抑制蛋白突变体 与P-Rh* 和Rh* 都具有高亲和力的结合物将用于研究 棒中信号关闭和恢复的动力学。几种先天性视力 疾病与视杆细胞中过量的视紫红质信号有关。 具有增强的关闭能力的组成型活性抑制蛋白突变体 这种信号传导似乎是这些疾病的基因治疗的逻辑工具。 这些突变体的治疗潜力将在这些模型中进行测试。 特别是在表达缺乏视紫红质的视紫红质的小鼠中 激酶磷酸化位点，并在视紫红质激酶敲除小鼠中， 抑制蛋白的代偿性变化是否能使他们的反应正常化 动力学和预防光依赖性视网膜变性。

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.