Desensitization of Cone Visual Signaling Pathways

 描述(申请人提供)：脊椎动物视杆和视锥中的光感受器信号由一系列精确计时的事件组成，这些事件对光感受器在广泛的光强度范围内发挥作用至关重要。虽然圆锥体在暗光下工作，在强光下很容易饱和，但圆锥体对光不那么敏感，恢复得更快，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(GRKs)分别通过视紫红质和视锥视蛋白的磷酸化，启动视杆细胞和视锥细胞光反应的关闭。我们已经发现，包括人类在内的许多脊椎动物都在视锥细胞中表达GRK1和GRK7，这引发了一个问题：这些激酶在光反应和适应中是否具有截然不同或重叠的功能？我们还发现，在暗适应的动物中，这两种酶都被cAMP依赖的蛋白激酶(PKA)磷酸化，在光适应的动物中去磷酸化。在体外，磷酸化会降低这些GRK的活性。因此，我们提出了一个新的假说，即cAMP通过其下游的PKA在光转导和/或适应中发挥重要的调节作用。我们已经建立了模型动物来(A)确定GRK1和GRK7在活体视锥细胞中所扮演的不同或重叠的角色，以及(B)检验cAMP介导的视杆细胞和视锥细胞中这些激酶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阐述了GRK1和GRK7对利用转基因斑马鱼进行锥体光反应和适应的贡献。与老鼠不同，斑马鱼在锥体中同时表达GRK1和GRK7，与人类相似。因此，斑马鱼是研究锥体的最佳遗传模型。我们使用TALEN干扰grk1a和grk7b的基因，以创造零突变。通过对5DPF斑马鱼的视网膜电信号(ERG)的分析，我们可以确定每一种激酶对锥体信号的贡献。在这个发育阶段，斑马鱼的视网膜在功能上是一个“全视锥”视网膜。光的强度/反应 在有或没有背景光的情况下，系列和配对闪光实验将被用来确定单独删除这些激酶对光响应和适应的动力学的影响。特定目的2研究斑马鱼球果中PKA对GRK1和GRK7的磷酸化作用。我们已经构建了表达磷酸化位点突变体Grk1b-S21a、Grk1b-S21e、Grk7a-S33A和Grk7a-S33E的细胞系。这些突变斑马鱼品系将与基因敲除品系杂交，并通过与针对特定目的1描述的实验类似的实验进行评估。特定目的3解决了突变S21A和S21E已敲入Grk1基因的小鼠的视杆和视锥中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。吸入电极记录和ERG将被用来确定GRK1的磷酸化在小鼠视杆和视锥细胞的光感受器信号中的作用。

 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.