Regulation of the Endocytic Trafficking of CFTR

描述（由申请人提供）：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，以确定致死性疾病囊性纤维化（CF）的治疗靶点。CFTR在上皮细胞的顶端质膜中表达，在那里它作为cAMP激活的Cl-通道起作用。CFTR介导的跨极化上皮细胞的Cl-转运通过调节通道活性和通过控制质膜中CFTR通道的数量来调节。? F508，CF中最常见的突变，减少了质膜中CFTR通道的数量，因为？F508-CFTR不能有效地从内质网中输出，并且因为F508-CFTR的质膜半衰期较短。F508-CFTR降低。降低质膜半衰期的机制？F508-CFTR尚未完全理解，部分原因是促进CFTR在质膜上的内吞运输的蛋白质相互作用尚未完全阐明。在初步研究中，我们确定了几种蛋白质，调节贩运CFTR在人类气道上皮细胞的顶膜。阐明这些蛋白质在CFTR运输中的作用对于理解顶膜运输缺陷至关重要？F508-CFTR。因此，我们建议测试的假设，气道细胞顶端膜密度的WT-CFTR和？F508-CFTR受蛋白质相互作用的差异调节，所述蛋白质相互作用发生在它们从顶膜内化、沿内吞途径运输沿着以及分选用于再循环或降解的过程中。为了验证这一假设，我们提出了三个具体目标：具体目标#1。测试Dab 2通过促进气道上皮细胞中CFTR内吞作用来抑制顶膜中CFTR表达的假设。这个具体目标的目的是阐明Dab 2在CFTR内吞作用的作用，并检查是否？F508突变加速Dab 2介导的CFTR的内吞作用。具体目标#2检验c-Cbl通过促进气道上皮细胞中CFTR的内吞作用来抑制顶膜中CFTR的表达的假设。本研究的目的是阐明c-Cbl及其衔接蛋白CIN 85在CFTR内吞作用中的作用，并确定c-Cbl及其衔接蛋白CIN 85在CFTR内吞作用中的作用。F508突变加速c-Cbl介导的CFTR的内吞作用。具体目标#3通过对气道上皮细胞中溶酶体降解的内化CFTR进行分选，检验Rab 4抑制顶膜中CFTR表达的假设。这一具体目标的目标是阐明Rab 4在靶向内化CFTR降解中的作用，并确定Rab 4是否参与了CFTR的降解。F508突变加速Rab 4介导的内化CFTR的分选。我们预计，我们的研究，在人气道上皮细胞进行：（1）将扩大我们的理解的内吞贩运CFTR;（2）将阐明质膜半衰期减少的机制？F508-CFTR;（3）将为CF患者提供新的治疗方法。公共卫生相关性。囊性纤维化（CF）是一种遗传性疾病，在美国每2,500名出生的儿童中就有一名受到影响。这种疾病影响呼吸和消化，目前还没有治愈这种疾病的方法。CF患者不能将盐（氯化钠）移入和移出某些细胞，包括那些排列在肺和胰腺上的细胞，因此产生粘稠的粘液和其他分泌物。这项应用的长期目标是开发一种药物，可以恢复盐进出细胞的运动，减轻CF患者的症状。

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the endocytic trafficking pathways of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in order to identify a therapeutic target for the fatal disease, cystic fibrosis (CF). CFTR is expressed in the apical plasma membrane in epithelial cells where it functions as a cAMP-activated Cl- channel. CFTR mediated Cl- transport across polarized epithelial cells is regulated by modulating channel activity and by controlling the number of CFTR channels in the plasma membrane. ?F508, the most common mutation in CF, reduces the number of CFTR channels in the plasma membrane because ?F508-CFTR is not efficiently exported from the endoplasmic reticulum and because the plasma membrane half-life of ?F508-CFTR is reduced. The mechanism of reduced plasma membrane half-life of ?F508-CFTR is not completely understood, in part, because the protein interactions that facilitate the endocytic trafficking of CFTR at the plasma membrane have not been completely elucidated. In preliminary studies we identified several proteins that regulate trafficking of CFTR at the apical membrane in human airway epithelial cells. Elucidating the role of these proteins in CFTR trafficking will be critical for understanding the apical membrane trafficking defect of ?F508-CFTR. Accordingly, we propose to test the hypothesis that the airway cell apical membrane density of WT-CFTR and ?F508-CFTR is differentially regulated by protein interactions that occur during their internalization from the apical membrane, trafficking along the endocytic pathway, and sorting for either recycling or degradation. To test this hypothesis we propose three specific aims: Specific Aim #1. Test the hypothesis that Dab2 inhibits the expression of CFTR in the apical membrane by facilitating CFTR endocytosis in airway epithelial cells. The goal of this specific aim is to elucidate the role of Dab2 in CFTR endocytosis and to examine whether the ?F508 mutation accelerates the Dab2 mediated endocytosis of CFTR. Specific Aim #2. Test the hypothesis that c-Cbl inhibits the expression of CFTR in the apical membrane by facilitating CFTR endocytosis in airway epithelial cells. The goal of this specific aim is to elucidate the role of c-Cbl and its adaptor protein, CIN85 in CFTR endocytosis and to determine whether the ?F508 mutation accelerates the c-Cbl mediated endocytosis of CFTR. Specific Aim #3. Test the hypothesis that Rab4 inhibits the expression of CFTR in the apical membrane by sorting the internalized CFTR for lysosomal degradation in airway epithelial cells. The goal of this specific aim is to elucidate the role of Rab4 in targeting internalized CFTR for degradation and to determine whether the ?F508 mutation accelerates the Rab4 mediated sorting of internalized CFTR. We anticipate that our studies, performed in human airway epithelial cells: (1) will expand our understanding of the endocytic trafficking of CFTR; (2) will elucidate the mechanism of decreased plasma membrane half-life of ?F508-CFTR; and (3) will lead to a new therapeutic approach in patients with CF. PUBLIC HEALTH RELEVANCE. Cystic Fibrosis (CF) is an inherited disease that affects one in every 2,500 children born in the US. The disease affects breathing and digestion and there is currently no cure for the disease. CF patients cannot move salt (sodium chloride) into and out of certain cells, including those that line the lungs and pancreas and as a result produce thick, sticky mucus and other secretions. The long-term goal of this application is to develop a drug that will restore salt movement into and out of cells and alleviate the symptoms in CF patients.