Opsins, G Protein Pathways and Regulation in RPE Cells

描述(申请人提供)：脊椎动物的视蛋白在光感受器和视网膜色素上皮(RPE)在视觉过程中起着基本的作用。光感受器中的视色素与11顺式视网膜结合，负责视觉兴奋的启动。RPE中的视黄素样视黄样视蛋白与全反式视网膜结合，可能在生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，在光适应过程中对11顺式视网膜的正常合成和视紫红质的再生是必需的。假设RGR在11-顺式视网膜的光依赖合成和视紫红质的再生中起中心作用，因此RGR参与视觉周期。这项拨款申请的目的是通过在分子和生理水平上证明RGR功能的详细机制模型来证明这一假说。 RGR视蛋白的研究受到可获得性、低丰度、生化特性以及缺乏合适的细胞培养模型等方面的限制。为了能够研究RGR视蛋白，已经创建了三种实验方法来解决这一研究问题。从牛RPE中分离到一种具有功能的RGR视蛋白。第二，建立了稳定表达RGR并能处理维甲酸的RPE细胞培养模型。第三，已经产生了具有丰富表型的RGR视蛋白基因敲除小鼠。这些实验方法将在这项拨款提案中用于进一步研究RGR的功能及其在RPE中的维甲酸代谢和调节中的作用。 通过保持视紫红质水平和视网膜敏感性，RGR视蛋白基因有助于预防夜盲，并为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细胞如何工作具有重要意义，而RGR功能缺陷的表征可能会揭示人类RPE细胞是如何经历功能障碍和恶化导致疾病的。由于RGR参与基本的视觉过程，RGR的缺陷很可能损害RPE和视网膜的健康。在常染色体显性或隐性视网膜色素变性患者中，人类RGR基因突变与视网膜色素变性分离，表明RGR视蛋白对神经视网膜的健康和活性的重要性。光感受器或RPE细胞中的视黄醇代谢错误可能会导致A2E水平异常，A2E是一种主要的脂褐素化合物，在衰老和老年性黄斑变性期间在RPE中积累。

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.