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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们的长期目标是阐明囊性纤维化跨膜传导调节因子（CFTR）的内吞运输途径，以开发一种策略来纠正囊性纤维化（CF）个体中deltaF 508-CFTR的缺陷性内吞运输。CF中最常见的突变Δ F508导致：（1）CFTR在内质网（ER）中的保留，（2）CFTR Cl通道的开放概率降低，和（3）CFTR的质膜半衰期降低。因此，由Δ F508突变引起的缺陷的校正将需要组合疗法，其包括：（1）增加的ER退出（2）增加的Cl通道活性，和（3）增加的质膜半衰期。在这些点中，关于调节delta 508-CFTR的质膜半衰期的机制尤其知之甚少。在该提议中待测试的假设是，与wt-CFTR相比，deltaF 508-CFTR的短质膜半衰期是由deltaF 508-CFTR的内吞运输的调节改变引起的。为了验证这一假设，我们提出了三个具体目标：具体目标#1。检验以下假设：与wt-CFTR相比，deltaF 508-CFTR的短质膜半衰期是由deltaF 508-CFTR的改变的内吞运输引起的。该特定目的的目标是确定deltaF 508-CFTR的质膜半衰期降低是否由CFTR的加速内吞作用或减弱的内吞再循环引起。具体目标#2测试Rab 5a和Rab 4调节CFTR的内吞运输的假设。该特定目的的目标是确定这些蛋白质是否调节CFTR的内吞运输以及Rab 5a和Rab 4的改变的表达是否是deltaF 508-CFTR的短质膜半衰期的原因。具体目标#3为了表征Rab 5a和Rab 4与CFTR之间的相互作用。这个特定目标的目的是确定Rab 5a和/或Rab 4是否直接与CFTR相互作用，deltaF 508突变是否改变Rab 5a和Rab 4与CFTR之间的结合亲和力，并鉴定Rab 5a和Rab 4相互作用的蛋白质，其可能调节CFTR的内吞运输。我们预期这些研究将阐明控制deltaF 508-CFTR的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CF和其他常见疾病的新的治疗策略，类似于CF，导致蛋白质运输的异常调节。

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.