Function and Regulation of PKA-mediated GRK Phosphorylation in Photoreceptors

描述（由申请人提供）：脊椎动物视杆细胞和视锥细胞的光传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下发挥作用所必需的。我们已经确定视网膜特异性G蛋白偶联受体激酶GRK1和GRK7在视杆细胞和视锥细胞的恢复和适应中起关键作用，它们在体外和体内都是camp依赖性蛋白激酶（PKA）的底物。在体外，PKA的磷酸化降低了这些激酶磷酸化底物视蛋白的能力。人类视锥细胞同时表达GRK1和GRK7，而小鼠则缺乏GRK7基因，在视锥细胞中只表达GRK1。这两种激酶都与人类视网膜病变有关，如Oguchi病、静止性夜盲症综合征和增强型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状况。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定义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（dpf）的功能上是一个“全锥体”视网膜，在锥体中表达Grk1和Grk7。为了评估PKA磷酸化的影响，将产生转基因鱼，表达磷酸化位点被消除的突变体（Ser到Ala）和磷酸化被带负电荷的氨基酸模仿的突变体（Ser到Glu）。这些鱼类的野生型蛋白将被morpholinos抑制，并通过视网膜电图（ERG）分析检测突变体的影响。我们的研究小组最近表明，腺苷酸环化酶1 （Adcy1）基因的磷酸化水平在零中受到影响，而在转导蛋白（Tr?）敲除小鼠中不受影响。为了评估ppa介导的磷酸化在锥体富集的幼体斑马鱼中的调节作用，我们将研究缺乏锥体转导素（Tc?）的非斑马鱼和内源性Adcy1被morpholinos抑制的野生型斑马鱼。Grk1和Grk7磷酸化水平将通过免疫印迹和免疫细胞化学分析确定，并通过ERG分析测量视觉功能。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。

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. 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. As cAMP is regulated by light in the vertebrate retina, it may regulate the light response of cones via mechanisms not previously described. It is anticipated that these studies will lead to a better understanding of cone function and advance our ability to prevent cone-related diseases.