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很好的模型系统，因为它是一个定义良好的层状神经元组织，非常容易接受各种实验操作。此外，视网膜有密切相关的神经细胞类别，视锥和视杆感受器，它们在形态和功能上相似，但在许多尚不清楚的分子组成和控制其功能的机制上不同。光感受器是高度极化和分隔的细胞。它们代表了适应于捕获和处理光子刺激的细胞的缩影。虽然许多光感受器成分在光激活的级联反应中的作用已经知道很多，但对于这些成分是如何被正确地处理并以矢量的方式分类到光感受器的外节亚细胞室的了解很少。特别是，七种跨膜光受体Opsins的加工、分选和功能表达的分子机制仍有待剖析。事实上，这一信息对所有七种跨膜受体都是缺乏的。视蛋白约占光感受器外节膜蛋白总量的90%。这些细胞可能表达一种高效的生物发生机制，以功能性地表达由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种跨膜受体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视蛋白生物发生生物学过程中潜在的分子组成和机制。如果想要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，这也是重要的。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的组成部分--亲环素相关蛋白，它选择性地陪伴并增强长波长视觉受体的功能产生。这种新的成分与多域蛋白RanBP2相同。这项建议的总体重点是从分子上剖析RanBP2及其调控分子组件在光感受器功能中的作用，特别是在颜色视蛋白的生物发生中的作用。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。这项提案将解决这些具体问题。1.红光蛋白中对识别RanBP2的RBD4和CY-PPlase结构域具有重要作用的分子决定因素是什么？2.RanBP2的其余初级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产生中有什么作用？

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?