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PIGMENT DEPHOSPHORYLATION IN MAMMALIAN ROD AND CONE PHOTORECEPTORS

哺乳动物视杆细胞和视锥细胞光感受器中的色素脱磷酸化

基本信息

批准号：
9756392
负责人：
Vladimir Jivkov Kefalov
金额：
$ 36.23万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9756392
关键词：
Address Affect Animals Arrestins Biochemical Biochemical Reaction Cone Dark Adaptation Disease Enterobacteria phage P1 Cre recombinase Enzymes Expression Profiling G-Protein-Coupled Receptors GRK1 gene GTP-Binding Proteins Kinetics Knockout Mice Light Light Adaptations Link Mediating Molecular Morphology Mus Mutant Strains Mice Natural regeneration Night Blindness Opsin Patients Phosphoric Monoester Hydrolases Phosphorylation Photophobia Photoreceptors Phototransduction Pigments Play Predisposition Process Protein Dephosphorylation Protein Subunits Protein phosphatase Proteins Rattus Reaction Retinal Cone Retinal Degeneration Retinal Pigments Rhodopsin Role Signal Transduction Signaling Protein Testing Therapeutic Time Vertebrate Photoreceptors Vision Disorders experimental study in vivo in vivo evaluation novel photoactivation photoreceptor degeneration response retinal rods visual cycle

项目摘要

ABSTRACT The state of phosphorylation mediates the activity of many G protein-coupled receptors. In photoreceptors, the phosphorylation of the visual pigment has been well characterized. However, an often-overlooked step in the resetting of the visual pigment following its photoactivation is its dephosphorylation. Despite remarkable progress in understanding G protein signaling in general, and phototransduction in particular, the enzyme responsible for catalyzing this reaction in vivo remains unknown. Also unknown is the role of visual pigment dephosphorylation in modulating the kinetics of dark adaptation or the susceptibility of photoreceptors to light damage. We have generated mice with loxP-flanked catalytic α-subunit of PP2A (Ppp2cafl/fl) which will allow us to investigate the role of this enzyme in pigment dephosphorylation and the function and survival of mammalian photoreceptors. Crossing these animals with mice expressing Cre recombinase selectively in rod or cone photoreceptors has allowed us to generate rod- and cone-specific PP2A Cα knockout mice. We will perform experiments to determine whether pigment dephosphorylation is suppressed in these mice. These experiments will establish whether PP2A is the elusive pigment phosphatase in mammalian rods and cones. We will also determine how the deletion of PP2A Cα affects the expression profile, morphology, and survival of photoreceptors. We will also analyze the light responses of PP2A Cα-deficient mouse rods and cones to determine the role of PP2A in modulating phototransduction. By in vivo ERG recordings, we will also determine whether the deletion of PP2A affects the kinetics of photoreceptor pigment regeneration and dark adaptation. These experiments will establish the role of pigment dephosphorylation in regulating the dark adaptation of mammalian rods and cones. The link between pigment phosphorylation and photoreceptor degeneration will also be explored using a combination on mutant mice lacking arrestin, rhodopsin kinase, and PP2A. Finally, we will also test the hypothesis that light-induced phosphorylation of ground state cone visual pigment reduces the overall light sensitivity and represents a novel mechanism for mammalian cone background light adaptation. Collectively, our experiments will establish the molecular mechanism for visual pigment dephosphorylation in mammalian photoreceptors. They will also determine the role of pigment dephosphorylation in controlling the kinetics of mammalian rod and cone dark adaptation, as well as the therapeutic potential of modulating this enzymatic reaction in light- or opsin-induced retinal degeneration.

摘要磷酸化状态介导许多G蛋白偶联受体的活性。在光感受器中，视色素的磷酸化已得到很好的表征。然而，一个经常被忽视的步骤，视色素在其光活化之后的重置是其去磷酸化。尽管引人注目在理解G蛋白信号传导的一般进展，特别是光转导，酶在体内催化这种反应的原因仍然未知。同样未知的是视觉色素的作用调节暗适应动力学或光感受器对光的敏感性中的去磷酸化损害我们已经产生了具有loxP侧翼的PP 2A催化α亚基（Ppp 2cafl/fl）的小鼠，这将使我们能够研究这种酶在色素去磷酸化中的作用，哺乳动物的光感受器将这些动物与在杆中选择性表达Cre重组酶的小鼠杂交或视锥光感受器使我们能够产生视杆细胞和视锥细胞特异性PP 2A Cα基因敲除小鼠。我们将进行实验以确定这些小鼠中的色素去磷酸化是否受到抑制。这些实验将确定PP 2A是否是哺乳动物视杆细胞和视锥细胞中难以捉摸的色素磷酸酶。我们还将确定PP 2AC α的缺失如何影响细胞的表达谱、形态和存活。光感受器我们还将分析PP 2A Cα缺陷小鼠视杆细胞和视锥细胞的光反应，确定PP 2A在调节光转导中的作用。通过在体ERG记录，我们还将确定 PP 2A的缺失是否影响光感受器色素再生和暗适应的动力学。这些实验将确定色素去磷酸化在调节黑色素细胞暗适应中的作用。哺乳动物的视杆细胞和视锥细胞。色素磷酸化和感光细胞退化之间的联系将也可以使用缺乏抑制蛋白、视紫红质激酶和PP 2A的突变小鼠的组合进行探索。最后我们还将检验光诱导的基态视锥细胞色素磷酸化降低视锥细胞色素水平的假设。整体光敏感性，并代表了一种新的机制，哺乳动物锥背景光适应。总的来说，我们的实验将建立视觉色素去磷酸化的分子机制，哺乳动物的光感受器他们还将确定色素去磷酸化在控制细胞凋亡中的作用。哺乳动物视杆细胞和视锥细胞暗适应的动力学，以及调节这种适应的治疗潜力。光或视蛋白诱导的视网膜变性中的酶促反应。