Function and Regulation of PKA-mediated GRK Phosphorylation in Photoreceptors

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。斑马鱼的视网膜在受精后4-7天是一个“全视锥”视网膜，在视锥细胞中表达Grk1和Grk7。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。通过视网膜电信号(ERG)分析，这些鱼类中的野生型蛋白将被吗啉抑制，并检测突变体的影响。我们的团队最近发现，腺酰环化酶1(Adcy1)基因的磷酸化水平受到零影响，而转导蛋白(Tr？)不受影响。基因敲除老鼠。为了评估PKA介导的磷酸化在富含锥体的斑马鱼幼体中的调节作用，我们将研究缺乏锥体转导蛋白(TC？)的斑马鱼Nof。以及野生型斑马鱼，其中内源性Adcy1已被吗啉抑制。Grk1和Grk7的磷酸化水平将通过免疫印迹和免疫细胞化学分析来确定，视功能的测量将通过ERG分析来进行。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。

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 two kinases in regards to 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. 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 electroretinogram (ERG) analysis. Our group has recently shown that levels of phosphorylation are affected in null for the adenylyl cyclase 1 (Adcy1) gene and are unaffected in transducin (Tr?) knockout mice. To evaluate the regulation of PKA-mediated phosphorylation in the cone-enriched larval zebrafish, we will examine nof zebrafish that lack cone transducin (Tc?) and wildtype zebrafish in which endogenous Adcy1 has been suppressed with morpholinos. Levels of Grk1 and Grk7 phosphorylation will be determined by immunoblot and immunocytochemical analysis, and visual function measured by ERG analysis. These studies will provide a foundation for understanding the novel role of phosphorylation by PKA on recovery and adaptation in vertebrate cones.