Opsins, G Protein Pathways and Regulation in RPE Cells

RPE 细胞中的视蛋白、G 蛋白通路和调节

• 批准号: 6546678
• 负责人: HENRY K FONG
• 金额: $ 38.25万
• 依托单位: DOHENY EYE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6546678
• 关键词: G protein; affinity chromatography; alcohol oxidoreductases; all trans retinol; binding proteins; cis trans isomerization; cone cell; electroretinography; gene targeting; genetically modified animals; laboratory mouse; laboratory rabbit; nicotinamide adenine dinucleotide; protein protein interaction; protein structure function; receptor coupling; receptor expression; recombinant proteins; retinal pigment epithelium; retinaldehyde; rhodopsin; rod cell; tissue /cell culture; transfection; visual phototransduction

DESCRIPTION (provided by applicant): Vertebrate opsins in photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11 -cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and may play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) in the RPE and Muller cells is an opsin that is necessary for normal synthesis of 11 -cis-retinal and regeneration of rhodopsin during light adaptation. It is hypothesized that RGR has a central role in light-dependent synthesis of 11-cis-retinal and the regeneration of rhodopsin, hence RGR is involved in the visual cycle. The objective of this grant application is to prove this hypothesis by demonstration of a detailed mechanistic model for the function of RGR at the molecular and physiological levels. The study of RGR opsin is impeded by limitations in accessibility, low abundance, biochemical properties, and the lack of an appropriate cell culture model. To be able to study the RGR opsin, three experimental approaches to this research problem have been created. A functional RGR opsin from bovine RPE has been isolated. Second, a cell culture model of RPE cells that stably express RGR and remain able to process retinoids has been established. Third, the RGR opsin knockout mouse with an informative phenotype has been produced. These experimental approaches will be used in this grant proposal to further investigate the function of RGR and its role in retinoid metabolism and regulation in the RPE. By preserving rhodopsin levels and retinal sensitivity, the RGR opsin gene helps to prevent night blindness and provides a selective advantage for species subject to wide variation in environmental luminance. Further understanding of RGR at the biochemical level will be important in learning how RPE cells work, and characterization of defects in RGR function may show how human RPE cells undergo dysfunction and deteriorate leading to disease. Since RGR is involved in a fundamental visual process, defects in RGR are likely to impair the health of the RPE and retina. The importance of RGR opsin to the health and viability of the neuroretina is shown by mutations in the human RGR gene that segregate with retinitis pigmentosa (RP) in patients with autosomal dominant or recessive RP. Errors in retinoid metabolism in either the photoreceptors or RPE cells may lead to abnormal levels of A2E, a major compound of lipofuscin that accumulates in the RPE during aging and age-related macular degeneration.

描述（由申请人提供）：光感受器和视网膜色素上皮（RPE）中的脊椎动物视蛋白在视觉过程中具有重要作用。光感受器中的视觉色素与 11-顺式视网膜结合，负责启动视觉兴奋。 RPE 中的类视网膜色素视蛋白与全反式视网膜结合，可能在发色团代谢中发挥重要作用。 RPE 和 Muller 细胞中的视网膜 G 蛋白偶联受体 (RGR) 是一种视蛋白，对于光适应过程中 11-顺式视黄醛的正常合成和视紫红质的再生是必需的。据推测，RGR 在 11-顺式视黄醛的光依赖性合成和视紫红质的再生中起核心作用，因此 RGR 参与视觉循环。本次拨款申请的目的是通过在分子和生理水平上演示 RGR 功能的详细机制模型来证明这一假设。 RGR 视蛋白的研究受到可及性、丰度低、生化特性的限制以及缺乏适当的细胞培养模型的阻碍。为了能够研究 RGR 视蛋白，针对该研究问题创建了三种实验方法。从牛 RPE 中分离出功能性 RGR 视蛋白。其次，建立了稳定表达 RGR 并保持处理类视黄醇能力的 RPE 细胞的细胞培养模型。第三，具有信息表型的 RGR 视蛋白敲除小鼠已经生产出来。这些实验方法将在本拨款提案中使用，以进一步研究 RGR 的功能及其在 RPE 中类视黄醇代谢和调节中的作用。 通过保持视紫质水平和视网膜敏感性，RGR 视蛋白基因有助于预防夜盲症，并为受环境亮度变化较大的物种提供选择优势。在生化水平上进一步了解 RGR 对于了解 RPE 细胞如何工作非常重要，RGR 功能缺陷的表征可能表明人类 RPE 细胞如何发生功能障碍和恶化导致疾病。由于 RGR 参与基本的视觉过程，因此 RGR 缺陷可能会损害 RPE 和视网膜的健康。 RGR 视蛋白对神经视网膜健康和活力的重要性通过人类 RGR 基因的突变得以体现，该基因与常染色体显性或隐性 RP 患者的视网膜色素变性 (RP) 分离。光感受器或 RPE 细胞中的类视黄醇代谢错误可能导致 A2E 水平异常，A2E 是脂褐质的主要化合物，在衰老和年龄相关性黄斑变性期间在 RPE 中积累。