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: 7712221
• 负责人: Ellen Ruth Weiss
• 金额: $ 18.5万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7712221
• 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）：脊椎动物杆和锥体的光转导由一系列精确的定时事件组成，这些事件对于光感受器在不断变化的光条件的环境中起作用。我们已经确定，视网膜特异性G蛋白偶联受体激酶GRK1和GRK7在杆和锥体中的恢复和适应中起着关键作用，都是cAMP依赖性蛋白激酶（PKA）在体外和体内的cAMP依赖性蛋白激酶（PKA）。 PKA磷酸化降低了这些激酶在体外磷酸化其底物的能力。与小鼠不同，人锥同时表达GRK1和GRK7，它们缺少GRK7的基因，并且仅在锥中表达GRK1。两种激酶都与人类视网膜病有关，例如Oguchi病，固定的夜失明综合征和增强的S锥综合征。因此，了解其体内调节将有助于理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，因此我们建议使用斑马鱼幼虫作为模型来定义PKA的GRK1和GRK7磷酸化的功能后果。斑马鱼视网膜在施肥后4-7天（DPF）的功能上是全孔视网膜，并在锥体中表达GRK1和GRK7。形态学将用于抑制GRK1，GRK7或激酶的表达，以及通过光震和电视图（ERG）分析评估的结果。 GRK1和GRK7的相对水平将以从斑马鱼锥形蛋白启动子控制的斑马鱼中分离出来的圆锥体中进行测量。为了评估PKA磷酸化的影响，将产生转基因鱼类的表达突变体，其中消除了磷酸化位点（SER至ALA）和突变体，其中磷酸化被带负电荷的氨基酸模拟的突变体（Sermoin Aid）（ser ser ser ser to Glu）。这些鱼类中的形态蛋白将抑制野生型蛋白质以及通过光酶分析和ERG测量的突变体的作用。这些研究将为理解PKA磷酸化在脊椎动物锥中恢复和适应的新作用提供基础。 公共卫生相关性：锥体与杆的敏感性和动力学不同，以及它们对遗传和环境诱导的疾病过程的敏感性。我们已经确定GRK1和GRK7被CAMP依赖性蛋白激酶磷酸化。由于cAMP受脊椎动物视网膜中的光调节，因此可以通过先前未描述的机制来调节锥对光的响应。预计这些研究将使人们对锥体功能有更好的了解提高了我们预防锥体相关疾病的能力。