DARTMOUTH COL COBRE: P2: REGULATION OF ENDOCYTIC TRAFFICKING OF CFTR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们的长期目标是阐明囊性纤维化跨膜电导调节器(CFTR)的胞内转运途径，以便开发一种策略来纠正囊性纤维化(CF)患者DeltaF508-CFTR的缺陷胞内转运。Cf中最常见的突变DeltaF508导致：(1)CFTR滞留在内质网(ER)；(2)CFTRCI通道开放概率降低；(3)CFTR质膜半衰期缩短。因此，纠正由deltaF508突变引起的缺陷将需要综合治疗，其中包括：(1)增加内质网退出；(2)增加CI通道活性；(3)增加质膜半衰期。在这些点中，特别是对调节delta508-CFTR质膜半衰期的机制知之甚少。本提案中要检验的假设是，与wt-CFTR相比，deltaF508-CFTR的质膜半衰期较短，这是由于deltaF508-CFTR对内吞转运的调节发生了变化。为了检验这一假说，我们提出了三个特定的目标：特定的目标#1.检验如下假设，即与wt-CFTR相比，deltaF508-CFTR的质膜半衰期较短是由于deltaF508-CFTR的内吞转运改变所致。这一特定目的的目的是确定deltaF508-CFTR质膜半衰期的减少是由CFTR的内吞作用加速还是内吞作用减弱引起的。特定目的#2.检验Rab5a和Rab4调控CFTR胞内转运的假设。这一特定目的的目的是确定这些蛋白是否调控CFTR的内吞运输，以及Rab5a和Rab4的表达变化是否与deltaF508-CFTR较短的质膜半衰期有关。具体目的#3.研究Rab5a和Rab4与CFTR的相互作用。其目的是确定Rab5a和/或Rab4是否直接与CFTR相互作用，deltaF508突变是否改变了Rab5a与Rab4和CFTR之间的结合亲和力，并鉴定可能调节CFTR内吞转运的Rab5a和Rab4相互作用的蛋白。我们预计这些研究将阐明控制deltaF508-CFTR质膜半衰期的细胞机制。此外，我们预计，了解这些机制将导致针对CF和其他常见疾病的新治疗策略，这些疾病类似于CF，是由蛋白质运输的异常调节引起的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-term goal is to elucidate the endocytic trafficking pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) in order to develop a strategy to correct defective endocytic trafficking of deltaF508-CFTR in individuals with Cystic Fibrosis (CF). DeltaF508, the most common mutation in CF causes: (1) retention of CFTR in the endoplasmic reticulum (ER), (2) reduced open probability of the CFTR CI channel, and (3) decreased plasma membrane half-life of CFTR. Thus, correction of the defects caused by the deltaF508 mutation will require a combination therapy that includes: (1) increased exit from the ER (2) increased CI channel activity, and (3) increased plasma membrane half-life. Of these points, particularly little is known about the mechanisms that regulate the plasma membrane half-life of delta508-CFTR. The hypothesis to be tested in this proposal is that the short plasma membrane half-life of deltaF508-CFTR compared to wt-CFTR results from altered regulation of endocytic trafficking of deltaF508-CFTR. To test this hypothesis we propose three specific aims: Specific Aim #1. Test the hypothesis that the short plasma membrane half-life of deltaF508-CFTR compared to wt-CFTR results from altered endocytic trafficking of deltaF508-CFTR. The goal of this specific aim is to determine whether the decreased plasma membrane half-life of deltaF508-CFTR is caused by accelerated endocytosis or attenuated endocytic recycling of CFTR. Specific Aim #2. Test the hypothesis that Rab5a and Rab4 regulate endocytic trafficking of CFTR. The goals of this specific aim are to determine whether these proteins regulate the endocytic trafficking of CFTR and whether altered expression of Rab5a and Rab4 is responsible for the short plasma membrane half-life of deltaF508-CFTR. Specific Aim #3. To characterize the interactions between Rab5a and Rab4 with CFTR. The goals of this specific aim are to determine if Rab5a and or Rab4 interact directly with CFTR, whether the deltaF508 mutation alters the binding affinities between the Rab5a and Rab4 and CFTR, and to identify the Rab5a and Rab4 interacting proteins that may regulate the endocytic trafficking of CFTR. We anticipate that these studies will elucidate the cellular mechanisms that control the plasma membrane half-life of deltaF508-CFTR. Furthermore, we anticipate that understanding these mechanisms will lead to novel therapeutic strategies for CF and other common diseases that, similar to CF, result from abnormal regulation of protein trafficking.