STRUCTURE/FUNCTION ANALYSIS OF RANBP2 IN THE NEURORETINA

脊椎动物视网膜是研究基本神经生物学过程的一个极好的模型系统，因为它是一个定义良好的分层神经元组织，高度适应各种实验操作。此外，视网膜有密切相关的神经细胞类别，视锥细胞和视杆细胞，它们在形态和功能上相似，但在许多未知的分子成分和控制其功能的机制上不同。光感受器是高度极化和区隔化的细胞。它们代表了适应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甚多，但对这些成分如何以矢量方式正确地加工和分类到光感受器的外段亚细胞区室知之甚少。特别是，7种跨膜光受体视蛋白的加工、分类和功能表达的分子机制仍有待深入研究。事实上，所有7种跨膜受体都缺乏这一信息。视蛋白约占光感受器外节膜蛋白总量的90%。这些细胞可能表达了一种高效的生物机制来功能性地表达大量的视蛋白，这些视蛋白是由这些细胞不断产生的。因此，光感受器也是研究七种跨膜感受器生物发生的一个很好的模型系统。我们的长期目标是确定光感受器细胞中视蛋白生物发生的生物学过程的分子成分和机制。如果想了解由视蛋白突变引起的遗传性视网膜病变的分子发病机制，这一点也很重要。最近，我们已经从分子上鉴定并部分表征了光感受器机制的一个新组成部分，即一种亲环蛋白相关蛋白，它选择性地陪伴并增强了长波视觉受体的功能产生。这种新成分与多结构域蛋白RanBP2相同。本提案的总体重点是分子解剖RanBP2及其调节分子组装在光感受器功能中的作用，特别是在色视蛋白的生物发生中。为此，我们将采用分子、生化、遗传和细胞生物学技术的多学科方法。这项建议将处理这些具体问题。1. 红视蛋白中的哪些分子决定因素对RBD4和RanBP2的CY-PPlase结构域的识别很重要？2. RanBP2其余主要结构模块的功能是什么？3. RanBP2在光感受器中红绿色素的生理生成中有什么作用？

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?