STRUCTURE/FUNCTION ANALYSIS OF RANBP2 IN THE NEURORETINA

神经视网膜中 RANBP2 的结构/功能分析

• 批准号: 6384717
• 负责人: PAULO A FERREIRA
• 金额: $ 25.07万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384717
• 关键词: SDS polyacrylamide gel electrophoresis; cone cell; gene mutation; genetically modified animals; immunoprecipitation; laboratory mouse; mass spectrometry; molecular chaperones; molecular shape; peptidylprolyl isomerase; protein binding; protein biosynthesis; protein folding; protein protein interaction; protein structure function; protein transport; retina; rhodopsin; rod cell; visual photoreceptor; visual pigments; yeast two hybrid system

The vertebrate retina is an excellent model system to study basic neurobiological processes because it is a well defined stratified neuronal tissue that is highly amenable to a wide variety of experimental manipulations. Moreover, the retina has closely related classes of neuronal cells, the cone and rod photoreceptors, which are morphologically and functionally alike, but otherwise distinct in many, yet unknown, molecular components and mechanisms governing their function. Photoreceptors are highly polarized and compartmentalized cells. They represent the epitome of a cell adapted to capture and process photon stimuli. Although a great deal is known about the role of many photoreceptor components in the light- activated cascade, very little is understood on how these components are correctly processed and sorted, in a vectorial fashion, to the outer segment subcellular compartment of photoreceptors. In particular, the molecular mechanisms underlying the processing, sorting and functional expression of the seven-transmembrane light-receptors, opsins, remains to be dissected. Indeed, this information is lacking for all seven-transmembrane receptors. Opsin constitutes about 90% of the total membrane protein in the outer segments of photoreceptors. These cells likely express a highly efficient biogenic machinery to functionally express the vast amounts of opsin that are continuously produced by these cells. Thus, photoreceptors are also an excellent model systems to study the biogenesis of seven-transmembrane receptors. Our long term goal is to identify the molecular components and mechanisms underlying the biological processes of opsin biogenesis in photoreceptor cells. This is also important if one wants to understand the molecular pathogenesis of inherited retinopathies caused by mutations in opsins. Recently, we have molecularly identified and partially characterized a novel component of the photoreceptor machinery, a cyclophilin-related protein, that selectively chaperones and enhances the functional production of long wave length visual receptors. This novel component is identical to the multi-domain protein, RanBP2. The overall focus of this proposal is to molecularly dissect the role of RanBP2 and its modulating molecular assemblies in phtoreceptor function and, in particular, in biogenesis of color opsins. To this end, we will undertake a multi-disciplinary approach of molecular, biochemical, genetic and cell biology techniques. This proposal will address these specific questions. 1. What are the molecular determinants in red opsin important for the recognition of RBD4 ane CY-PPlase domains of RanBP2? 2. What is the function of the remaining primary structural modules of RanBP2? 3. What are the effects of RanBP2 in the physiological production of red- green pigment in photoreceptors?

脊椎动物视网膜是研究基本神经生物学过程的优秀模型系统，因为它是一种明确的分层神经元组织，非常适合各种实验操作。此外，视网膜具有密切相关的神经元细胞类别，即视锥细胞和视杆细胞感光细胞，它们在形态和功能上相似，但在许多但未知的分子成分和控制其功能的机制方面有所不同。光感受器是高度极化和区室化的细胞。 它们代表了适合捕获和处理光子刺激的细胞的缩影。尽管人们对许多光感受器成分在光激活级联中的作用了解很多，但对于如何以矢量方式正确处理和分类这些成分到光感受器的外段亚细胞区室的了解却很少。特别是，七次跨膜光受体视蛋白的加工、分类和功能表达背后的分子机制仍有待剖析。事实上，所有七次跨膜受体都缺乏这一信息。视蛋白约占光感受器外节总膜蛋白的 90%。这些细胞可能表达高效的生物机制，以功能性地表达这些细胞持续产生的大量视蛋白。因此，光感受器也是研究七次跨膜受体生物发生的优秀模型系统。我们的长期目标是确定感光细胞中视蛋白生物发生的生物过程的分子成分和机制。如果想了解视蛋白突变引起的遗传性视网膜病的分子发病机制，这一点也很重要。最近，我们从分子上鉴定并部分表征了光感受器机制的一种新成分，即亲环蛋白相关蛋白，它选择性地陪伴并增强长波长视觉受体的功能产生。这种新成分与多结构域蛋白 RanBP2 相同。该提案的总体重点是从分子角度剖析 RanBP2 及其调节分子组装体在光感受器功能中的作用，特别是在颜色视蛋白的生物合成中的作用。为此，我们将采用分子、生物化学、遗传和细胞生物学技术的多学科方法。该提案将解决这些具体问题。 1. 红视蛋白中对于识别 RanBP2 的 RBD4 ane CY-PPlase 结构域很重要的分子决定因素是什么？ 2. RanBP2其余主要结构模块的作用是什么？ 3. RanBP2对光感受器红绿色素的生理产生有什么影响？