Desensitization of Cone Visual Signaling Pathways

视锥细胞视觉信号通路的脱敏

• 批准号: 9334886
• 负责人: Ellen Ruth Weiss
• 金额: $ 38万
• 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 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9334886
• 关键词: Address; Amino Acids; Animal Model; Animals; Biochemical; Blindness; Burn injury; Cessation of life; Collaborations; Color Visions; Cone; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Darkness; Development; Disease; Electrodes; Electroretinography; Event; Fertilization; Fishes; G protein coupled receptor kinase; GRK1 gene; GRK7 gene; Genes; Genetic Models; Human; In Vitro; Individual; Kinetics; Knock-out; Larva; Lead; Light; Mammals; Mediating; Methods; Modeling; Mus; Mutate; Mutation; Opsin; Pathology; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photoreceptors; Phototransduction; Physiological; Physiology; Play; Process; Protein Kinase; Proteins; Recovery; Retina; Retinal; Retinal Cone; Rhodopsin; Role; Series; Signal Pathway; Signal Transduction; Study models; Suction; Testing; Transgenic Organisms; Vertebrate Photoreceptors; Vision; Visual; Zebrafish; defined contribution; desensitization; design; disability; experimental study; homologous recombination; in vivo; light intensity; mutant; novel; prevent; public health relevance; receptor; response; retinal rods; transcription activator-like effector nucleases

 DESCRIPTION (provided by applicant): Photoreceptor signaling in vertebrate rods and cones consists of a series of precisely timed events that are critical for photoreceptors to function under a broad range of light intensities. While rods operate under dim light and are easily saturated in response to bright light, cones are less sensitive to light, recover more rapidly and are able to function under intense light. G protein-coupled receptor kinases (GRKs) in the vertebrate retina initiate turnoff of the photoresponse in both rods and cones via phosphorylation of rhodopsin and the cone opsins, respectively. We have discovered that many vertebrate species, including humans, express both GRK1 and GRK7 in cones, raising the question: do these kinases have distinct or overlapping functions in the photoresponse and adaptation? We have also shown that both kinases are phosphorylated by cAMP-dependent protein kinase (PKA) in dark-adapted animals and dephosphorylated in light-adapted animals. Phosphorylation reduces the activity of these GRKs in vitro. Therefore we propose a novel hypothesis that cAMP plays an important regulatory role in phototransduction and/or adaptation through its downstream kinase, PKA. We have generated model animals to (a) determine the distinct or overlapping roles played by GRK1 and GRK7 in cones in vivo and (b) test the hypothesis that cAMP-mediated phosphorylation of these kinases in rods and cones plays an important role in photoreceptor signaling. Specific Aim 1 addresses the contributions of GRK1 and GRK7 to the cone photoresponse and adaptation using genetically modified zebrafish. Unlike mice, zebrafish express both GRK1 and GRK7 in cones, similar to humans. Therefore zebrafish is the best genetic model for studies of cones. We used TALENs to disrupt the genes for grk1a and grk7b to create null mutants. Analyzing these lines by electroretinography (ERG) in 5 dpf zebrafish will allow us to define the contribution of each kinase to cone signaling. At this stage of development, the zebrafish retina is functionally an "all cones" retina. A light intensity/response series and paired flash experiments with or without background light will be used to determine the effect of deleting these kinases individually on the kinetics of the photoresponse and adaptation. Specific Aim 2 addresses the role of phosphorylation of GRK1 and GRK7 by PKA in zebrafish cones. We have generated lines expressing the phosphorylation-site mutants, Grk1b-S21A, Grk1b-S21E, Grk7a-S33A and Grk7a-S33E. These mutant zebrafish lines will be crossed with the knockout lines and evaluated using experiments similar to those described for Specific Aim 1. Specific Aim 3 addresses the influence of phosphorylation of GRK1 by PKA in both rods and cones in mice where mutations S21A and S21E have been knocked into the Grk1 gene. These genetically modified mice express levels of mutant GRK1 that are identical to the wild type protein. Suction electrode recording and ERG will be performed to define the role of phosphorylation of GRK1 on photoreceptor signaling in rods and cones in mice.

 描述（由申请人提供）：脊椎动物视杆细胞和视锥细胞中的光感受器信号传导由一系列精确定时的事件组成，这些事件对于光感受器在广泛的光强度下发挥作用至关重要。视杆细胞在昏暗的光线下工作，并且在强光下很容易饱和，而视锥细胞对光不太敏感，恢复得更快，并且能够在强光下发挥作用。脊椎动物视网膜中的 G 蛋白偶联受体激酶 (GRK) 分别通过视紫红质和视锥细胞视蛋白的磷酸化启动视杆细胞和视锥细胞中光响应的关闭。我们发现，包括人类在内的许多脊椎动物物种在视锥细胞中同时表达 GRK1 和 GRK7，这就提出了一个问题：这些激酶在光响应和适应中是否具有不同或重叠的功能？我们还表明，这两种激酶在暗适应动物中被 cAMP 依赖性蛋白激酶 (PKA) 磷酸化，而在光适应动物中被去磷酸化。磷酸化会降低这些 GRK 的体外活性。因此，我们提出了一个新的假设，即 cAMP 通过其下游激酶 PKA 在光转导和/或适应中发挥重要的调节作用。我们生成了模型动物来（a）确定GRK1和GRK7在体内视锥细胞中发挥的不同或重叠的作用，以及（b）测试cAMP介导的这些激酶在视杆细胞和视锥细胞中的磷酸化在光感受器信号转导中发挥重要作用的假设。具体目标 1 解决了 GRK1 和 GRK7 对转基因斑马鱼视锥细胞光响应和适应的贡献。与小鼠不同，斑马鱼在视锥细胞中同时表达 GRK1 和 GRK7，与人类相似。因此，斑马鱼是研究视锥细胞的最佳遗传模型。我们使用 TALEN 破坏 grk1a 和 grk7b 的基因来创建无效突变体。通过 5 dpf 斑马鱼的视网膜电图 (ERG) 分析这些线路，我们将能够定义每种激酶对视锥信号传导的贡献。在这个发育阶段，斑马鱼视网膜在功能上是“全视锥细胞”视网膜。光强度/响应 有或没有背景光的系列和配对闪光实验将用于确定单独删除这些激酶对光响应和适应动力学的影响。具体目标 2 阐述了 PKA 在斑马鱼视锥细胞中磷酸化 GRK1 和 GRK7 的作用。我们已经生成了表达磷酸化位点突变体 Grk1b-S21A、Grk1b-S21E、Grk7a-S33A 和 Grk7a-S33E 的品系。这些突变斑马鱼品系将与敲除品系杂交，并使用与特定目标 1 中所述类似的实验进行评估。特定目标 3 解决了 Grk1 基因中突变 S21A 和 S21E 已被敲入的小鼠视杆细胞和视锥细胞中 PKA 对 GRK1 磷酸化的影响。这些转基因小鼠表达的突变 GRK1 水平与野生型蛋白相同。将进行抽吸电极记录和 ERG，以确定 GRK1 磷酸化对小鼠视杆细胞和视锥细胞中光感受器信号传导的作用。