Opsins, G Protein Pathways and Regulation in RPE Cells

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基本作用。光感受器中的视色素与11 -顺式-视黄醛结合，并负责视觉兴奋的起始。视网膜色素样视蛋白与全反式视黄酸结合，可能在发色团代谢中起重要作用。视网膜G蛋白偶联受体（RGR）在视网膜色素上皮细胞和米勒细胞是一种视蛋白，是必要的正常合成的11 -顺式-视网膜和再生的视紫红质在光适应。据推测，RGR在光依赖性的11-顺式-视黄醛合成和视紫红质再生中具有中心作用，因此RGR参与视觉周期。本基金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设。 RGR视蛋白的研究受到可及性、低丰度、生物化学性质以及缺乏合适的细胞培养模型的限制。为了能够研究RGR视蛋白，已经创建了三种针对该研究问题的实验方法。已经从牛RPE中分离出功能性RGR视蛋白。第二，建立了稳定表达RGR并保持能够加工类维生素A的RPE细胞的细胞培养模型。第三，已经产生了具有信息表型的RGR视蛋白敲除小鼠。这些实验方法将用于本基金提案中，以进一步研究RGR的功能及其在RPE中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症，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细胞如何工作将是重要的，并且RGR功能缺陷的表征可以显示人类RPE细胞如何经历功能障碍并恶化导致疾病。由于RGR参与基本的视觉过程，RGR的缺陷可能会损害RPE和视网膜的健康。RGR视蛋白对神经视网膜的健康和活力的重要性通过在患有常染色体显性或隐性RP的患者中与视网膜色素变性（RP）分离的人RGR基因中的突变显示。光感受器或RPE细胞中类维生素A代谢的错误可能导致A2 E水平异常，A2 E是脂褐素的主要化合物，在衰老和年龄相关性黄斑变性期间在RPE中积累。

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.