Desensitization of Cone Visual Signaling Pathways

描述（由申请人提供）：视锥细胞负责日光视觉，仅占人类感光细胞群的5-6%。其余的细胞，即视杆细胞，调节昏暗的光线视觉。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知识大多来自对视杆细胞的研究。 已经观察到视杆细胞和视锥细胞对光的反应的敏感性和动力学的显著差异。视锥细胞对光线的敏感度低于视杆细胞，并且从光照中恢复的速度更快。在多个步骤，包括G蛋白激活，受体失活和下游信号事件的差异可能是部分负责杆和锥光转导之间观察到的差异。本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），GRK 7。GRK 7只在我们研究过的哺乳动物的视锥细胞中表达，除了在小鼠和大鼠中，它们在视锥细胞中表达GRK 1（视杆细胞GRK）。我们的实验室已经证明GRK 7磷酸化来自13线地松鼠（一种视锥占优势的哺乳动物）的完整视网膜中的视锥视蛋白，这表明GRK 7通过磷酸化参与视锥视蛋白的失活。由于GRK 7和GRK 1在人类视锥细胞中共表达，它们可能都在视锥细胞视蛋白失活中起作用，但可能受到不同的调节。这些酶的生化特性将在体外进行比较，并研究其活性中翻译后修饰的作用。将在两种动物模型中检查视锥视蛋白的原位磷酸化，即具有“全视锥”视网膜的Nrl-/-GRK 1-/-小鼠和非洲爪蟾（Xenopus laevis）（南非爪蛙）的富含视锥的视网膜。GRK 7将被引入Nrl-/-GRK 1-/-小鼠中以确定该激酶是否可以在用于磷酸化和电生理学研究的生化测定中替代GRK 1。还将GRK 7和GRK 1的PKA和自磷酸化位点突变体引入这些小鼠中并进行类似表征。我们已经发现爪蟾视网膜表达GRK 7和GRK 1。因此，我们将描述这2种激酶的细胞表达模式，并设计一种使用吗啉代靶向非洲爪蟾GRK 7的反义策略。使用这种“敲低”方法，我们计划建立一个模型，可用于确定GRK 7是否是脊椎动物锥细胞中正常光转导所必需的。将对视锥细胞进行ERG测量，以分析GRK“敲低的功能后果。"

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."