The Role of Phosphorylation in Photoreceptor Cell Biology

描述（由申请人提供）：脊椎动物视杆细胞和视锥细胞中的光转导由一系列精确定时的事件组成，这些事件是光感受器在不断变化的光条件的环境中发挥作用所必需的。我们已经确定，视网膜特异性G蛋白偶联受体激酶，GRK 1和GRK 7，这在恢复和适应杆和锥中发挥关键作用，都是cAMP依赖性蛋白激酶（PKA）在体外和体内的底物。PKA的磷酸化降低了这些激酶在体外磷酸化其底物视蛋白的能力。人类视锥细胞同时表达GRK 1和GRK 7，不像小鼠，小鼠缺乏GRK 7基因，只在视锥细胞中表达GRK 1。这两种激酶都与人类视网膜病有关，如Oguchi病、静止性夜盲综合征和增强型S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条件。由于小鼠不是研究这2种激酶在人类视觉中作用的合适模型，因此我们建议使用斑马鱼幼虫作为模型来定义PKA磷酸化GRK 1和GRK 7的功能后果。斑马鱼视网膜在受精后4-7天（dpf）时在功能上是全视锥视网膜，并且在视锥中表达GRK 1和GRK 7。将使用吗啉代抑制GRK 1、GRK 7或这两种激酶的表达，并通过视动和视网膜电图（ERG）分析评价结果。GRK 1和GRK 7的相对水平将在从在斑马鱼视锥细胞转导蛋白启动子的控制下表达EGFP的斑马鱼分离的视锥细胞中测量。为了评价PKA对磷酸化的影响，将产生表达磷酸化位点已被消除的突变体（Ser至Ala）和磷酸化被带负电荷的氨基酸模拟的突变体（Ser至Glu）的转基因鱼。这些鱼中的野生型蛋白质将被吗啡啉抑制，突变体的影响通过视动分析和ERG来测量。这些研究将为理解PKA磷酸化在脊椎动物视锥细胞恢复和适应中的新作用提供基础。 公共卫生相关性：视锥细胞与视杆细胞在光反应的敏感性和动力学以及它们对遗传和环境诱导的疾病过程的易感性方面不同。我们已经确定，GRK 1和GRK 7磷酸化cAMP依赖性蛋白激酶。由于cAMP在脊椎动物视网膜中受光调节，因此它可以通过先前未描述的机制调节视锥细胞对光的反应。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.