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（由适用提供）：脊椎动物棒和锥体中的光感受器信号传导由一系列精确的定时事件组成，这些事件对于光感受器在广泛的光强度下起作用至关重要。当杆在昏暗的光线下运行，并且响应明亮的光线而易于饱和，但锥体对光的敏感性较小，恢复更快，并且能够在强光下运行。 G蛋白偶联受体激酶（GRKS）在脊椎动物视网膜中引发了偏离，我们发现许多脊椎动物（包括人类）在锥中表达GRK1和GRK7在锥中，提出了一个问题：这些激酶是否具有光孔索斯之间的独特或重叠的功能？我们还表明，两种激酶都被黑暗适应性动物中的cAMP依赖性蛋白激酶（PKA）磷酸化，并在灯光适应的动物中脱磷酸化。磷酸化在体外降低了这些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/或适应中起重要的调节作用。我们已经生成了模型动物，以（a）确定grk1和grk7在体内圆锥体中扮演的明显或重叠的作用，（b）检验以下假说：cAMP介导的这些激酶在杆和锥体中在光感受器信号传导中起重要作用。特定的目标1解决了GRK1和GRK7对锥形光响应和使用转基因斑马鱼适应的贡献。与小鼠不同，斑马鱼在锥体中同时表达GRK1和GRK7，类似于人类。因此，斑马鱼是锥研究研究的最佳遗传模型。我们使用Talens破坏了GRK1A和GRK7B的基因，以创建无效突变体。通过5 dpf斑马鱼中的电视图（ERG）分析这些线，将使我们能够定义每个激酶对锥形信号的贡献。在开发的这个阶段，斑马鱼视网膜在功能上是“全锥”视网膜。光强度/响应带有或没有背景光的串联和配对的闪光实验将用于确定分别删除这些激酶对光响应和适应动力学的效果。具体目标2解决了斑马鱼锥中PKA对GRK1和GRK7磷酸化的作用。我们生成了表达磷酸化位点突变体的线，GRK1B-S21A，GRK1B-S21E，GRK7A-S33A和GRK7A-S33E。这些突变的斑马鱼线将与敲除线交叉，并使用类似于针对特定目标1的实验进行评估。特定的目标3解决了PKA在杆和锥中PKA在突变S21A和S21E中的GRK1磷酸化的影响。这些一般修饰的小鼠表达与野生型蛋白相同的突变体GRK1水平。将执行吸力电极记录和ERG，以定义GRK1的光磷酸化在小鼠棒和锥中光感受器信号传导中的作用。