Function and Regulation of PKA-mediated GRK Phosphorylation in Photoreceptors

PKA 介导的光感受器 GRK 磷酸化的功能和调节

• 批准号: 8397943
• 负责人: Jared David Chrispell
• 金额: $ 5.22万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8397943
• 关键词: Affect; Amino Acids; Biological Assay; Calmodulin; Canis familiaris; Charge; Color Visions; Contrast Sensitivity; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dark Adaptation; Disease; Electroretinography; Embryo; Environment; Event; Family suidae; Fertility; Fertilization; Fishes; Foundations; G protein coupled receptor kinase; GRK1 gene; GRK7 gene; Genes; Human; Immunoblotting; In Vitro; Kinetics; Knockout Mice; Laboratories; Larva; Lead; Learning; Light; Light Adaptations; Measures; Mediating; Modeling; Mus; Night Blindness; Oligonucleotides; Opsin; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photoreceptors; Phototransduction; Play; Predisposition; Process; Protein Dephosphorylation; Protein Kinase; Proteins; Recovery; Regulation; Retina; Retinal Cone; Retinal Diseases; Retinal Pigments; Rhodopsin; Role; Series; Signal Pathway; Signal Transduction; Study models; Syndrome; System; Techniques; Testing; Time; Transducin; Transgenic Organisms; Variant; Vertebrate Photoreceptors; Vertebrates; Vision; Visual; Zebrafish; adenylyl cyclase 1; in vivo; mutant; novel; overexpression; prevent; promoter; response; retinal rods; visual adaptation; visual stimulus

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.

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两种激酶对人类视觉作用的合适模型，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。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。野生型蛋白质将被这些鱼中的吗啉抑制，并且通过视网膜电图（ERG）分析测量突变体的影响。我们的小组最近表明，磷酸化水平的影响，在空的腺苷酸环化酶1（Adcy 1）基因和不受影响的转导（Tr？）敲除小鼠为了评估PKA介导的磷酸化调节的锥丰富的斑马鱼幼虫，我们将研究nof斑马鱼缺乏锥转导（Tc？）和野生型斑马鱼，其中内源性Adcy 1已被吗啉代抑制。将通过免疫印迹和免疫细胞化学分析确定Grk 1和Grk 7磷酸化水平，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。 公共卫生关系：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的光响应。预计这些研究将导致更好地了解视锥细胞的功能，并提高我们预防视锥细胞相关疾病的能力。