Rhodopsin Trafficking and Retinal Degenerations

描述（由申请人提供）：分子神经科学和细胞生物学中最基本的问题之一是信号转导膜的正确组装，包括蛋白质成分的运输和分类。神经退行性疾病和其他遗传性疾病的主要原因是受体和其他信号或运输蛋白的不适当定位。本研究的目的是鉴定在运输过程中与视紫红质相互作用的蛋白和参与杆细胞外节盘膜生物形成的蛋白，然后确定视紫红质与其他蛋白的分子相互作用导致健康光感受器盘膜形成的分子机制。这项工作将进一步了解由受体和其他膜蛋白的不适当运输引起的神经退行性疾病的机制。该研究的重点是了解当视紫红质在其羧基端缺乏适当的结构时，蛋白质-蛋白质相互作用是有缺陷的，就像几种最严重的常染色体显性视网膜色素变性的情况一样。我们将使用我和我的同事开发的强大的鼠标敲入模型，以及这里提出的新模型。在Aim 1中，我们将通过亲和捕获实验，利用具有缺陷的羧基末端敲入动物的纯合子视紫红质突变体的视网膜提取物，鉴定与视紫红质羧基末端相互作用的蛋白质，以介导适当的运输和盘膜组装。在目的2中，我们将首先在体外，然后在体内描述一种突变的视紫红质Ter349Glu，它含有一个羧基末端延伸，导致视紫红质介导的常染色体显性视网膜色素变性的最严重形式之一。在Aim 3中，我们将开发一种新的工具，将人视紫红质融合到光激活的绿色荧光蛋白上，然后重复视紫红质羧基末端（rho-paGFP- 1D4）。这种结构将以两种不同的方式使用：首先，我们将验证一个假设，即在敲入动物的健康杆状体中，一个通畅的视紫红质羧基末端足以形成适当的外段。其次，我们将研究GFP光激活后特定蛋白-蛋白相互作用在视紫红质运输中的作用，使我们能够首次跟踪细胞中视紫红质亚群的运动。这为未来的体内贩运研究奠定了基础。公共卫生相关性：本研究的重点是了解当昏暗的光感受器视紫红质在其羧基端缺乏适当的结构时，蛋白质-蛋白质相互作用是有缺陷的，就像几种最严重的常染色体显性视网膜色素变性的情况一样。我们将研究视紫红质在正常杆状细胞形成和退化中的作用，并监测其贩运以更好地了解这些过程。

DESCRIPTION (provided by applicant): One of the most fundamental problems in molecular neuroscience and cell biology is the proper assembly of signal-transducing membranes including the transport and sorting of protein components. A major cause of neurodegenerative and other inherited disorders is the improper localization of receptors and other signaling or transport proteins. The goal of this study is to identify proteins that interact with rhodopsin during transport and those involved in the biogenesis of disk membranes in the outer segment of rod cells, and then determine the molecular mechanisms by which the molecular interactions of rhodopsin with other proteins lead to formation of healthy photoreceptor disk membranes. This work will further the understanding of the mechanisms of neurodegenerative disorders caused by improper trafficking of receptors and other membrane proteins. The focus of the proposed research is to understand protein-protein interactions that are defective when rhodopsin lacks the proper structure at its carboxy-terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will use powerful mouse knock-in models that my co-workers and I have developed, as well as new models proposed herein. In Aim 1, we will identify proteins that interact with rhodopsin's carboxy-terminus to mediate proper transport and disk membrane assembly through affinity-capture experiments using retinal extracts from homozygote rhodopsin mutants with defective carboxyl-termini knock-in animals. In Aim 2, we will characterize, first in vitro, then in vivo, a mutant rhodopsin, Ter349Glu, containing a carboxyl-terminal extension that causes one of the most severe forms of rhodopsin-mediated autosomal dominant retinitis pigmentosa. In Aim 3, we will develop a new tool, human rhodopsin fused to photoactivatable green fluorescent protein that is followed by a repeat of rhodopsin's carboxyl terminus (rho-paGFP- 1D4). This construct will be used in two distinct ways: first, we will test the hypothesis that an unobstructed rhodopsin carboxy-terminus is sufficient to form proper outer segments in healthy rods in knock-in animals. Second, we will study the role of specific protein-protein interactions in rhodopsin trafficking after photoactivation of GFP, enabling us to track the movement of subpopulations of rhodopsin in cells for the first time. This sets the stage for in vivo trafficking studies in the future. PUBLIC HEALTH RELEVANCE: The focus of this study is to understand protein-protein interactions that are defective when the dim light photoreceptor rhodopsin lacks the proper structure at its carboxy- terminus, as is the case in several of the most severe forms of autosomal dominant retinitis pigmentosa. We will study the role of rhodopsin in proper rod cell formation and degeneration, and monitor its trafficking to better understand these processes.