MOLECULAR BASIS OF PROTEIN TRANSPORT IN PHOTORECEPTORS

光感受器中蛋白质运输的分子基础

• 批准号: 2020030
• 负责人: CHING-HWA SUNG
• 金额: $ 21.74万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-01-31 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2020030
• 关键词: MDCK cell; Urodela; binding proteins; biological signal transduction; gene expression; gene mutation; genetically modified animals; laboratory mouse; nucleic acid sequence; organ culture; protein transport; retina degeneration; retinitis pigmentosa; rhodopsin; rod cell; visual photoreceptor; yeast two hybrid system

DESCRIPTION (Adapted from applicant's abstract): A newly independent applicant plans to continue work on mechanisms underlying the defects in rhodopsin mutations. Earlier work by the applicant established two different types of defects in rhodopsin mutants implicated in ADRP. One class is defective in protein exit from the ER/Golgi complex; the second class is incapable of polarized sorting and fails to targeting the rhodopsin to the rod outer segment. In this proposal, the applicant will follow up the observations on this second class of mutants in hopes of deciphering the mechanisms involved in rhodopsin localization. In the first specific aim, three complementary systems will be used to identify the signals responsible for the targeting of rhodopsin to the appropriate membrane. A polarized epithelial cell from dog kidney, the MDCK cell, will be used to analyze large numbers of rhodopsin mutants. To determine the suitability of this system for reflecting photoreceptor behavior, a salamander primary retinal culture will be examined for suitability as an experimental system. Finally, transgenic mice will be used to determine the reliability of both experimental systems in reflecting the in vivo situation. The work will establish the requirement and the sufficiency of the C-terminus domain as well as search for other important sequences in the sorting process. In the second specific aim, other molecular components of the sorting process will be sought by identifying proteins that bind to rhodopsin sorting sequences. Both the yeast two hybrid system and a direct protein/protein filter binding assays will be used. Identified genes will be sequenced to determine the nature of the encoded gene and their subcellular location will be determined.

描述（改编自申请人的摘要）：新独立 申请人计划继续处理缺陷的基础机制 视紫红质突变。申请人的早期工作建立了两个 Rhodopsin突变体中的不同类型的缺陷与ADRP有关。一 类别从ER/Golgi复合物退出蛋白质中有缺陷；第二个 班级无法进行两极分化的分类，并且无法针对 视紫红质到杆外段。在此提案中，申请人将 跟进对第二类突变体的观察，希望 解解参与视紫红质定位的机制。 在第一个特定目的中，将使用三个互补系统 确定负责将视紫红质靶向靶向的信号 适当的膜。狗肾脏的两极化上皮细胞， MDCK细胞将用于分析大量的视紫红质突变体。 确定该系统对于反射感光器的适用性 行为，将检查Salamander的原发性视网膜培养 作为实验系统的适用性。最后，转基因小鼠将是 用于确定两个实验系统在 反映体内情况。这项工作将确定 C-terminus域的需求和充分性 在排序过程中搜索其他重要序列。 在第二个特定目的中，排序的其他分子成分 通过识别与视紫红质结合的蛋白质来寻求过程 排序序列。酵母两个混合动力系统和直接 将使用蛋白质/蛋白质过滤器结合测定法。确定的基因 将测序以确定编码基因及其的性质 将确定亚细胞位置。