Desensitization of Cone Visual Signaling Pathways

视锥细胞视觉信号通路的脱敏

• 批准号: 7171761
• 负责人: Ellen Ruth Weiss
• 金额: $ 35.32万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-07 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171761
• 关键词: Desensitization; Cone; Visual; Signaling; Pathways

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."

描述（由申请人提供）：锥细胞负责日光视觉，仅占人类感光细胞群的5-6％。其余细胞，杆细胞介导昏暗的光视觉。由于大多数哺乳动物的锥体很少，因此我们大多数关于光转导的知识都来自对杆的研究。 在对光的反应的灵敏度和动力学中，棒和锥之间存在显着差异。锥体对光的敏感性不如杆敏感，并且从光照暴露中恢复的速度更快。多个步骤的差异，包括G蛋白激活，受体失活和下游信号传导事件可能部分造成观察到的杆和锥形光转导的差异。我们的实验室克隆了一种新型的，视网膜特异性的G蛋白偶联受体激酶（GRK），GRK7。 GRK7仅在我们检查的哺乳动物中仅表示，但在小鼠和大鼠中除外，而小鼠和大鼠除外，而锥子中的Grk1（杆细胞GRK）在锥体中。我们的实验室表明，GRK7磷酸化的锥蛋白在13层的地松鼠（一种圆锥体哺乳动物）中的完整视网膜中磷酸化，这表明GRK7参与磷酸化的锥蛋白蛋白。由于GRK7和GRK1在人锥中共表达，因此它们都可能在Opsin失活中发挥作用，但可能会有所不同。这些酶的生化特性将在体外进行比较，并在研究其活性中的作用。锥蛋白原位的磷酸化将在两种动物模型中检查NRL - / - grk1 - / - 小鼠，该模型具有“全锥”视网膜，以及Xenopus laevis的圆锥视网膜，南非爪状的青蛙。 GRK7将引入NRL - / - GRK1 - / - 小鼠，以确定该激酶是否可以在生化测定中代替GRK1进行磷酸化和电生理研究。 GRK7和GRK1的PKA和自磷酸化位点突变体也将引入这些小鼠并类似地表征。我们发现Xenopus视网膜Express GRK7和GRK1。因此，我们将表征这两种激酶的细胞表达模式，并使用形态剂在Xenopus中靶向GRK7。使用这种“敲低”方法，我们计划建立一个模型，该模型可用于确定GRK7是否对于脊椎动物锥的正常光转导至关重要。将对锥体进行ERG测量，以分析GRK“敲低”的功能后果。