The Role of Phosphorylation in Photoreceptor Cell Biology

磷酸化在感光细胞生物学中的作用

• 批准号: 7904076
• 负责人: Ellen Ruth Weiss
• 金额: $ 21.98万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904076
• 关键词: Adult; Affect; African; Amino Acids; Animals; Antibodies; Behavioral Assay; Biological Assay; Cells; Cellular biology; Charge; Contrast Sensitivity; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dark Adaptation; Disease; Embryo; Environment; Event; Family suidae; Fertilization; Fishes; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; G protein coupled receptor kinase; GRK; GRK1 gene; GRK7 gene; Genes; Human; Immunoblotting; Immunofluorescence Microscopy; In Vitro; Injection of therapeutic agent; Kinetics; Larva; Lead; Light; Light Adaptations; Measures; Modeling; Mus; Night Blindness; Opsin; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photoreceptors; Phototransduction; Play; Predisposition; Primates; Process; Protein Dephosphorylation; Protein Kinase; Proteins; Rana; Recovery; Regulation; Relative (related person); Retina; Retinal Cone; Retinal Diseases; Rhodopsin; Role; Series; Signal Transduction; Staging; Study models; Syndrome; Testing; Time; Transducin; Transgenic Organisms; Vertebrate Photoreceptors; Vertebrates; Vision; Visual; Xenopus laevis; Xenopus sp.; Zebrafish; behavior test; in vivo; light intensity; mutant; novel; overexpression; prevent; promoter; public health relevance; response; retinal rods

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.

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照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 GRK1 和 GRK7 在视杆细胞和视锥细胞的恢复和适应中发挥着关键作用，它们都是体外和体内 cAMP 依赖性蛋白激酶 (PKA) 的底物。 PKA 磷酸化会降低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 GRK1 和 GRK7，而小鼠则缺乏 GRK7 基因，视锥细胞只表达 GRK1。这两种激酶都与人类视网膜病有关，例如大口病、静止性夜盲症和增强型 S 锥综合症。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 2 种激酶在人类视觉中的作用的合适模型，因此我们建议使用斑马鱼幼虫作为模型来定义 PKA 磷酸化 GRK1 和 GRK7 的功能后果。受精后 4-7 天 (dpf)，斑马鱼视网膜在功能上是全视锥细胞视网膜，并在视锥细胞中表达 GRK1 和 GRK7。吗啡啉将用于抑制 GRK1、GRK7 或两种激酶的表达，并通过光动和视网膜电图 (ERG) 分析评估结果。 GRK1 和 GRK7 的相对水平将在斑马鱼视锥细胞转导蛋白启动子控制下从表达 EGFP 的斑马鱼分离的视锥细胞中进行测量。为了评估 PKA 磷酸化的影响，将产生转基因鱼，表达磷酸化位点已被消除的突变体（Ser 至 Ala）和带负电荷氨基酸模拟磷酸化的突变体（Ser 至 Glu）。这些鱼中的吗啡啉会抑制野生型蛋白质，并且通过光动分析和 ERG 测量突变体的影响。这些研究将为理解 PKA 磷酸化对脊椎动物视锥细胞恢复和适应的新作用奠定基础。 公共健康相关性：视锥细胞与视杆细胞的不同之处在于光反应的敏感性和动力学，以及它们对遗传和环境诱发的疾病过程的易感性。我们已经确定 GRK1 和 GRK7 被 cAMP 依赖性蛋白激酶磷酸化。由于脊椎动物视网膜中的 cAMP 受光调节，因此它可能通过以前未描述过的机制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。