The Role of Phosphorylation in Photoreceptor Cell Biology

描述(申请人提供)：脊椎动物视杆和视锥中的光传导由一系列精确计时的事件组成，这些事件是光感受器在不断变化的光条件下发挥作用所必需的。我们已经确定，视网膜特异的G蛋白偶联受体激酶GRK1和GRK7在视杆细胞和视锥细胞的恢复和适应中发挥关键作用，在体外和体内都是cAMP依赖的蛋白激酶(PKA)的底物。在体外，PKA的磷酸化降低了这些酶对其底物--视蛋白的磷酸化能力。人的视锥细胞同时表达GRK1和GRK7，不像小鼠，小鼠缺乏GRK7基因，只在视锥细胞中表达GRK1。这两种激酶都与人类视网膜病变有关，如小口病、静止性夜盲综合征和增强型S视锥综合征。因此，了解它们在体内的调节将有助于了解视网膜的病理状态。由于小鼠不是研究这两种激酶在人类视觉中的作用的合适模型，我们建议使用斑马鱼幼虫作为模型来确定PKA磷酸化GRK1和GRK7的功能后果。斑马鱼视网膜是受精后4-7天的全视锥视网膜，在视锥细胞中表达GRK1和GRK7。Morpholinos将被用来抑制GRK1和/或GRK7的表达，并通过视动和视网膜电图(ERG)分析来评估结果。在斑马鱼锥体转导蛋白启动子的控制下，将在表达绿色荧光蛋白的斑马鱼中分离出GRK1和GRK7的相对水平。为了评估PKA磷酸化的影响，将产生表达磷酸化位点已被消除的突变体(Ser到Ala)和被带负电荷的氨基酸模拟磷酸化的突变体(Ser到Glu)的转基因FISH。这些鱼类中的野生型蛋白将被吗啉抑制，并通过光动力分析和ERG测量突变体的影响。这些研究将为理解PKA的磷酸化在脊椎动物圆锥体的恢复和适应中的新作用奠定基础。 公共卫生相关性：在光反应的敏感度和动力学以及对遗传和环境引起的疾病过程的敏感性方面，锥体不同于杆状细胞。我们已经确定GRK1和GRK7被cAMP依赖的蛋白激酶磷酸化。由于cAMP在脊椎动物视网膜中受光的调节，它可能通过先前未描述的机制来调节视锥细胞对光的反应。预计这些研究将有助于更好地了解视锥细胞的功能，提高我们预防视锥细胞相关疾病的能力。

DESCRIPTION (provided by applicant): Phototransduction in vertebrate rods and cones consists of a series of precisely timed events that are necessary for photoreceptors to function in an environment of continually changing light conditions. We have determined that the retina-specific G protein-coupled receptor kinases, GRK1 and GRK7, which play critical roles in recovery and adaptation in rods and cones, are both substrates for cAMP-dependent protein kinase (PKA) in vitro and in vivo. Phosphorylation by PKA reduces the ability of these kinases to phosphorylate their substrates, the opsins, in vitro. Human cones express both GRK1 and GRK7, unlike mice, which lack the gene for GRK7 and express only GRK1 in cones. Both kinases have been implicated in human retinopathies, such as Oguchi disease, a stationary night blindness syndrome, and Enhanced S Cone Syndrome. Therefore, understanding their regulation in vivo will contribute to the understanding of pathological conditions in the retina. Since mice are not an appropriate model for studying the role of these 2 kinases in human vision, we propose to use zebrafish larvae as a model to define the functional consequences of GRK1 and GRK7 phosphorylation by PKA. The zebrafish retina is functionally an all-cone retina at 4-7 days post fertilization (dpf) and expresses both GRK1 and GRK7 in cones. Morpholinos will be used to suppress the expression of GRK1, GRK7 or both kinases and the results evaluated by optokinetic and electroretinographic (ERG) analyses. The relative levels of GRK1 and GRK7 will be measured in cones isolated from zebrafish expressing EGFP under the control of the zebrafish cone transducin promoter. To evaluate the influence of phosphorylation by PKA, transgenic fish will be generated expressing mutants in which the phosphorylation sites have been eliminated (Ser to Ala) and mutants in which phosphorylation is mimicked by a negatively charged amino acid (Ser to Glu). The wild-type proteins will be suppressed by morpholinos in these fish and the effects of the mutants measured by optokinetic analysis and ERG. These studies will provide a foundation for understanding the novel role of phosphorylation by PKA on recovery and adaptation in vertebrate cones. PUBLIC HEALTH RELEVANCE: Cones differ from rods in the sensitivity and kinetics of the light response as well as their susceptibility to genetically and environmentally induced disease processes. We have determined that GRK1 and GRK7 are phosphorylated by cAMP-dependent protein kinase. Since cAMP is regulated by light in the vertebrate retina, it may regulate the response of cones to light via mechanisms not previously described. It is anticipated that these studies will lead to a better understanding of cone function advance our ability to prevent cone-related diseases.