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

基本信息

批准号：
8527511
负责人：
Jared David Chrispell
金额：
$ 5.39万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/8527511
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

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

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项目摘要

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.

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力方面的作用的合适模型，因此我们建议使用斑马鱼幼虫作为定义PKA GRK1和GRK7磷酸化的功能后果的模型。斑马鱼视网膜在受精后4-7天（DPF）的功能上是“全锥”视网膜，并在锥体中表达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，以及通过电子图（ERG）分析测量的突变体的作用。我们的小组最近表明，腺苷环酶1（ADCY1）基因的磷酸化水平受到NULL的影响，并且在跨杜以生意（TR？）敲除小鼠中不受影响。为了评估富含锥体的幼虫斑马鱼中PKA介导的磷酸化的调节，我们将研究缺少锥形trandducin（TC？）和Wildtype Zebrafish的NOF斑马鱼，其中内源性ADCY1在哪些内源性adcy1被抑制了吗？ GRK1和GRK7磷酸化水平将由免疫印迹和免疫细胞化学分析以及通过ERG分析测量的视觉功能确定。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。