Structure-function studies of visual arrestin

细胞对持续刺激的反应性降低，通常被称为脱敏，是一种普遍的生物学现象。视觉放大级联(以及其他G蛋白偶联受体的信号)通过两步机制减弱：视紫红质激酶使光激活的视紫红质(Rh*)磷酸化，然后arrestin与光激活的磷酸化视紫红质(P-Rh*)结合。Arrestin结合终止转导蛋白介导的信号转导，在光感受器细胞的恢复中发挥重要作用。 本研究的主要目的是阐明视觉arrestin功能的精细分子机制如何转化为它与视紫红质的及时结合、随后与磷光蛋白的解离、它在光中移位到视杆外段以及在黑暗中它如何移动到视紫红质内段。利用定点突变、直接结合实验、Arrestin和视紫红质的自旋标记、EPR光谱和X射线结晶学，我们将鉴定参与它们相互作用的arrestin和视紫红质残基。我们将阐明紧密结合arrestin所需的视紫红质结合磷酸盐的数量，以及arrestin和视紫红质二聚体在它们相互作用中的作用。我们将使用定制设计 在转基因小鼠中表达arrestin突变体，以研究杆状细胞中信号关闭和恢复的动力学，arrestin自结合的生理作用及其光依赖易位的功能。一些先天性疾病与视紫红质信号过多有关。我们已经创造了与P-Rh*和Rh*具有高亲和力的磷酸化不依赖的“超级拦截素”结合，这似乎是这些疾病的基因治疗的合理工具。使用“超级阻滞剂”作为矫正反应工具的可行性 还将测试视紫红质磷酸化位点或视紫红质激酶丢失的小鼠模型的动力学和预防视网膜变性。

The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Visual amplification cascade (and the 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 arrestin binding to light-activated phosphorylated rhodopsin (P-Rh*). Arrestin binding terminates transducin-mediated signaling, playing an important role in the recovery of photoreceptor cells. The main objective of this proposal is to elucidate how the fine molecular mechanisms of visual arrestin function translate into its timely binding to rhodopsin, subsequent dissociation from phosphoopsin, its translocation into rod outer segment in the light and its movement to the inner segment in the dark. Using site-directed mutagenesis, direct binding assay, spin labeling of arrestin and rhodopsin followed by EPR spectroscopy, and X-ray crystallography we will identify arrestin and rhodopsin residues participating in their interaction. We will elucidate the number of rhodopsin-attached phosphates necessary for tight arrestin binding and the role of arrestin and rhodopsin dimerization in their interaction. We will use custom-designed arrestin mutants expressed in transgenic mice to study the kinetics of signal shut-off and recovery in rods, the physiological role of arrestin self-association and function of its light-dependent translocation. Several congenital disorders are associated with excessive rhodopsin signaling. We have created phosphorylation-independent "super-arrestins" binding with high affinity to P-Rh* and Rh*, that appear to be logical tools for gene therapy of these disorders. The feasibility of using "super-arrestins" as tools for correcting response kinetics and preventing retinal degeneration in mouse models with the loss of rhodopsin phosphorylation sites or rhodopsin kinase will be also tested.