Desensitization of Cone Visual Signaling Pathways

描述(申请人提供)：视锥细胞负责日光视觉，仅占人类感光细胞总数的5%-6%。剩下的细胞，即视杆细胞，调节着暗淡的光线视觉。因为大多数哺乳动物的视锥细胞很少，所以我们关于光传导的大部分知识都来自于对视杆的研究。在对光的反应的敏感性和动力学方面，视杆细胞和视锥细胞之间存在着显著的差异。锥形细胞对光的敏感度比杆状细胞低，从光照中恢复的速度更快。多个步骤的差异，包括G蛋白激活、受体失活和下游信号事件，可能是观察到视杆细胞和视锥细胞光转导差异的部分原因。我们实验室克隆了一种新的视网膜特异性G蛋白偶联受体激酶(GRK)，GRK7。GRK7只在我们研究过的哺乳动物的视锥细胞中表达，除了小鼠和大鼠，它们在视锥细胞中表达GRK1(杆状细胞GRK)。我们的实验室已经证明，GRK7在13层地松鼠的完整视网膜中磷酸化视锥视蛋白，这表明GRK7参与了通过磷酸化使视锥视蛋白失活的过程。由于GRK7和GRK1在人视锥细胞中共表达，它们可能都在视锥细胞视蛋白失活中发挥作用，但可能受到不同的调控。这些酶的生化特性将在体外进行比较，并研究翻译后修饰在其活性中的作用。将在两个动物模型中检测视锥视蛋白的原位磷酸化，NRL-/-GRK1-/-小鼠的视网膜为“全视锥”，非洲爪蛙的视锥丰富的视网膜为南非爪蛙。GRK7将被引入到NRL-/-GRK1-/-小鼠中，以确定该激酶是否可以在用于磷酸化和电生理研究的生化检测中替代GRK1。GRK7和GRK1的PKA和自磷酸化位点突变体也将被引入这些小鼠，并具有类似的特征。我们发现非洲爪哇视网膜表达GRK7和GRK1。因此，我们将研究这两种蛋白的细胞表达模式，并设计一种针对非洲爪哇GRK7的反义策略。使用这种“敲除”方法，我们计划建立一个模型，可以用来确定GRK7是否对脊椎动物锥体的正常光转导是必不可少的。将对锥体进行ERG测量，以分析GRK“击倒”的功能后果。

DESCRIPTION (provided by applicant): Cone cells are responsible for daylight vision, making up only 5-6% of the photoreceptor cell population in humans. The remaining cells, the rod cells, mediate dim light vision. Because the majority of mammals have very few cones, most of our knowledge regarding phototransduction comes from the study of rods. Significant differences between rods and cones have been observed in the sensitivity and kinetics of the response to light. Cones are less sensitive to light than rods and recovery from light exposure occurs more rapidly. Differences at multiple steps, including G protein activation, receptor deactivation and downstream signaling events may be partly responsible for the observed differences between rod and cone phototransduction. Our laboratory has cloned a novel, retina-specific G protein-coupled receptor kinase (GRK), GRK7. GRK7 is expressed exclusively in cones in mammals we have examined, except in mouse and rat, which, instead express GRK1 (the rod cell GRK) in cones. Our laboratory has demonstrated that GRK7 phosphorylates cone opsins in intact retinas from the 13-lined ground squirrel, a cone-dominant mammal, suggesting that GRK7 is involved in deactivation of cone opsin by phosphorylation. Since GRK7 and GRK1 are co-expressed in human cones, they may both play a role in cone opsin deactivation but may be regulated differently. The biochemical properties of these enzymes will be compared in vitro and the role of posttranslational modifications in their activity investigated. Phosphorylation of cone opsins in situ will be examined in two animal models, the Nrl-/-GRK1 -/- mouse, which has an "all cone" retina, and the cone-rich retina of Xenopus laevis, the South African clawed frog. GRK7 will be introduced into the Nrl-/-GRK1 -/- mice to determine whether this kinase can substitute for GRK1 in biochemical assays for phosphorylation and electrophysiological studies. PKA and autophosphorylation site mutants of GRK7 and GRK1 will also be introduced into these mice and similarly characterized. We have found that Xenopus retinas express GRK7 and GRK1. Therefore, we will characterize the cellular expression pattern for these 2 kinases and design an antisense strategy using morpholinos to target GRK7 in Xenopus. Using this "knockdown" approach, we plan to establish a model that can be used to determine whether GRK7 is essential for normal phototransduction in vertebrate cones. ERG measurements on cones will be performed to analyze the functional consequences of GRK "knockdown."