Desensitization of Cone Visual Signaling Pathways

视锥细胞视觉信号通路的脱敏

基本信息

批准号：
9762105
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
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-07 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9762105
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 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

项目摘要

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，而视杆细胞在昏暗的光线下工作并且很容易响应饱和。在强光下，视锥细胞对光不太敏感，恢复得更快，并且能够在强光下在脊椎动物视网膜中启动 G 蛋白偶联受体激酶 (GRK) 发挥作用。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：这些激酶是否具有不同或重叠。我们还表明，在暗适应动物中，这两种激酶均被 cAMP 依赖性蛋白激酶 (PKA) 磷酸化在光适应动物中，磷酸化会降低这些 GRK 的活性，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。 (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) 检验以下假设：cAMP 介导的这些激酶在视杆细胞和视锥细胞中的磷酸化起着重要作用具体目标 1 使用转基因斑马鱼解决了 GRK1 和 GRK7 对视锥细胞光响应和适应的贡献，与人类相似，斑马鱼在视锥细胞中表达 GRK1 和 GRK7。我们使用 TALEN 破坏 grk1a 和 grk7b 的基因，通过分析这些品系来创建无效突变体。 5 dpf 斑马鱼的视网膜电图 (ERG) 将使我们能够定义每种激酶对视锥细胞信号传导的贡献，在这个发育阶段，斑马鱼视网膜在功能上是一个“全视锥细胞”视网膜。使用或不使用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 解决了 PKA 在斑马鱼视锥细胞中磷酸化 GRK1 和 GRK7 的作用。已生成表达磷酸化位点突变体 Grk1b-S21A、Grk1b-S21E、Grk7a-S33A 和Grk7a-S33E。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 中描述的类似的评估实验。特定目标 3 解决突变 S21A 的小鼠视杆细胞和视锥细胞中 PKA 对 GRK1 磷酸化的影响。和 S21E 已被敲入 Grk1 基因中，这些转基因小鼠表达的突变 GRK1 水平与野生型蛋白相同。将进行以确定 GRK1 磷酸化对小鼠视杆细胞和视锥细胞光感受器信号传导的作用。