STRUCTURE/FUNCTION ANALYSIS OF RANBP2 IN THE NEURORETINA

脊椎动物视网膜是一个很好的模型系统，研究基本的神经生物学过程，因为它是一个明确的分层的神经元组织，是非常适合各种各样的实验操作。此外，视网膜具有密切相关的神经元细胞类别，视锥和视杆光感受器，它们在形态和功能上相似，但在许多尚不清楚的分子组分和控制其功能的机制方面不同。光感受器是高度极化和区室化的细胞。 它们代表了一个细胞的缩影，适合捕捉和处理光子刺激。尽管对许多光感受器组分在光激活级联中的作用了解很多，但对这些组分如何以矢量方式正确加工和分选到光感受器的外节亚细胞区室了解甚少。特别是，七跨膜光受体，视蛋白的加工，分选和功能表达的分子机制仍有待解剖。事实上，所有七跨膜受体都缺乏这种信息。视蛋白占光感受器外节中总膜蛋白的约90%。这些细胞可能表达高效的生物机制，以功能性地表达由这些细胞连续产生的大量视蛋白。因此，光感受器也是研究七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中视蛋白生物合成的生物学过程的分子组成和机制。如果人们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，这也是很重要的。最近，我们已经从分子水平上鉴定并部分表征了感光器机制的一种新组分，一种亲环蛋白相关蛋白，其选择性地伴侣并增强长波长视觉受体的功能性生产。这种新的组分与多结构域蛋白RanBP 2相同。该提案的总体重点是从分子上剖析RanBP 2及其调节分子组装体在光感受器功能中的作用，特别是在彩色视蛋白的生物发生中。为此，我们将采用分子、生物化学、遗传学和细胞生物学技术的多学科方法。本提案将解决这些具体问题。1.红色视蛋白中哪些分子决定簇对于RBD 4和RanBP 2的CY-PPase结构域的识别很重要？2. RanBP 2的其余主要结构模块的功能是什么？3. RanBP 2在光感受器中红-绿色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?