Functional Characterization of RGR-opsin in Retinal Muller Cells

视网膜 Muller 细胞中 RGR-视蛋白的功能表征

• 批准号: 8965466
• 负责人: GABRIEL H TRAVIS
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8965466
• 关键词: 11-cis-Retinol; All-Trans-Retinol; Back; Bathing; Biochemical; Biochemical Pathway; Biological; Cells; Cone; Confocal Microscopy; Consumption; Cultured Cells; Disease; Electroretinography; Endoplasmic Reticulum; Esters; Exhibits; Exposure to; Eye; G protein-coupled receptor RGR; Genes; Goals; Human; Hydrolysis; Inherited; Invertebrates; Isomerase; Knock-out; Knockout Mice; Knowledge; Left; Life; Light; Lipids; Liquid substance; Mammalian Cell; Mediating; Metabolism; Methods; Microscopy; Muller' s cell; Mus; Mutation; Natural regeneration; Neural Retina; Opsin; Phenotype; Photobleaching; Photons; Photophobia; Photoreceptors; Physiological; Pigments; Play; Process; Production; Proteins; Recovery; Regulation; Research; Retina; Retinal; Retinal Ganglion Cells; Retinal Pigments; Retinaldehyde; Retinitis Pigmentosa; Retinoids; Retinol dehydrogenase; Rhodopsin; Role; Structure of retinal pigment epithelium; Testing; Tissues; Vertebrate Photoreceptors; Visual; Vitamin A; Work; animal tissue; cell type; chromophore; interest; light effects; melanopsin; programs; protein distribution; public health relevance; retinal rods; sensor; tissue culture; two-photon; visual cycle; visual process; visual processing

 DESCRIPTION (provided by applicant): Capture of a photon by a rhodopsin or cone-opsin pigment in a photoreceptor isomerizes the 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which activates the pigment. Shortly afterwards, the all-trans-RAL dissociates, leaving behind light-insensitive apo-opsin. To regenerate the opsin pigment, the released all-trans-RAL must be converted back to 11-cis-RAL, which then recombines with apo-opsin. Synthesis of visual chromophore takes place in the retinal pigment epithelium (RPE) and Müller cells of the retina, both adjacent to photoreceptors. The biochemical pathways that mediate this conversion are different in RPE and Müller cells. RPE cells produce 11-cis-RAL at a slow rate, and are thought to provide chromophore for both rods and cones under dim light. Müller cells mainly produce the chromophore- precursor, 11-cis-retinol (11-cis-ROL), which is utilized by cones, but not rods, to synthesize 11-cis-RAL. In bright light, Müller cells turn-over retinoids at a much higher rate than do RPE cells. RGR-opsin is a non-visual opsin located in both RPE and Müller cells. Mice with a knock-out mutation in the rgr gene exhibit slow regeneration of visual pigments and accumulate retinyl esters in the RPE and retina. Retinyl esters are lipid-soluble storage forms of retinol (vitamin A). Preliminary studies suggest that RGR-opsin effects light-dependent mobilization of retinyl esters in the RPE. Nothing is known about the function of RGR-opsin in Müller cells, which is the subject of the current study. This project tests the broad hypothesis that RGR-opsin regulates the flow of visual retinoids between RPE and Müller cells in a light-dependent fashion. For example, by stimulating hydrolysis of retinyl esters in the RPE, RGR-opsin depletes substrate for the isomerase in RPE cells (Rpe65) while providing all-trans-ROL substrate for the isomerase in Müller cells (DES1). This study will be performed on cultured mammalian cells expressing selected retinoid-processing proteins, and ocular tissues from genetically modified mice. Experimental methods include liquid chromatographic analysis of retinoids, single- and two-photon confocal microscopy of animal tissues and cultured cells, and ex vivo electroretinography of live mouse eyecups and retina explants.

 描述（由申请人提供）：光感受器中视紫红质或视锥蛋白色素对光子的捕获将11-顺式-视黄醇（11-cis-RAL）发色团异构化为全反式-视黄醇（all-trans-RAL），其激活色素。不久之后，全反式RAL解离，留下对光不敏感的脱辅基视蛋白。为了再生视蛋白色素，释放的全反式RAL必须转化回11-顺式RAL，然后与脱辅基视蛋白重组。视觉发色团的合成发生在视网膜的视网膜色素上皮（RPE）和Müller细胞中，两者都邻近光感受器。介导这种转化的生化途径在RPE和Müller细胞中是不同的。RPE细胞以缓慢的速率产生11-cis-RAL，并且被认为在昏暗的光下为视杆和视锥两者提供发色团。米勒细胞主要产生发色团前体11-顺式-视黄醇（11-cis-ROL），其被视锥细胞而非视杆细胞利用以合成11-cis-RAL。在明亮 Müller细胞以比RPE细胞高得多的速率转换类维生素A。RGR-视蛋白是位于RPE和Müller细胞两者中的非视觉视蛋白。rgr基因敲除突变的小鼠表现出视色素再生缓慢，并在RPE和视网膜中积累视黄酯。视黄酯是视黄醇（维生素A）的脂溶性储存形式。初步研究表明，RGR-视蛋白影响RPE中视黄酯的光依赖性动员。目前还不知道RGR-视蛋白在Müller细胞中的功能，这是目前研究的主题。该项目测试了RGR-视蛋白以光依赖性方式调节RPE和Müller细胞之间视觉类视色素的流动的广泛假设。例如，通过刺激RPE中视黄酯的水解，RGR-视蛋白消耗RPE细胞中异构酶的底物（Rpe65），同时为Müller细胞中异构酶提供全反式ROL底物（DES1）。本研究将在表达选定的类维生素A加工蛋白的哺乳动物培养细胞和转基因小鼠的眼组织中进行。实验方法包括类维生素A的液相色谱分析，动物组织和培养细胞的单光子和双光子共聚焦显微镜，以及活小鼠眼杯和视网膜外植体的离体视网膜电图。