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剂（CFTR）的内吞转运途径，以确定致命疾病囊性纤维化（CF）的治疗靶点。CFTR在上皮细胞的顶质膜中表达，在那里它作为camp激活的Cl-通道发挥作用。CFTR介导的Cl-在极化上皮细胞间的转运是通过调节通道活性和控制质膜中CFTR通道的数量来调节的。？F508是CF中最常见的突变，它减少了质膜中CFTR通道的数量，因为？F508-CFTR不能有效地从内质网输出，因为质膜半衰期？F508-CFTR降低。？的质膜半衰期缩短机理F508-CFTR尚未被完全了解，部分原因是促进CFTR在质膜上的内噬运输的蛋白质相互作用尚未完全阐明。在初步研究中，我们发现了几种调节CFTR在人气道上皮细胞顶膜运输的蛋白质。阐明这些蛋白在CFTR运输中的作用对于理解F508-CFTR的顶膜运输缺陷至关重要。因此，我们提出验证WT-CFTR和？F508-CFTR受蛋白相互作用的差异调控，这些相互作用发生在它们从根尖膜内化、沿内噬途径运输以及分选回收或降解的过程中。为了验证这一假设，我们提出了三个具体目标：验证Dab2通过促进CFTR在气道上皮细胞内吞作用抑制CFTR在顶膜表达的假设。这个特定目的的目的是阐明Dab2在CFTR内吞作用中的作用，并检查？F508突变加速了Dab2介导的CFTR内吞作用。具体目标2。验证c-Cbl通过促进CFTR在气道上皮细胞内吞作用抑制CFTR在顶膜表达的假设。本研究的目的是阐明c-Cbl及其接头蛋白CIN85在CFTR内吞作用中的作用，并确定？F508突变加速了c-Cbl介导的CFTR内吞作用。具体目标#3。通过对内化CFTR在气道上皮细胞中溶酶体降解的分选，验证Rab4抑制CFTR在顶膜表达的假设。本特定目的的目的是阐明Rab4在靶向内化CFTR降解中的作用，并确定？F508突变加速了Rab4介导的内化CFTR的分选。我们期望我们在人气道上皮细胞中进行的研究：(1)将扩大我们对CFTR内吞运输的理解；(2)阐明？F508-CFTR降低质膜半衰期的机理；(3)将为CF患者提供一种新的治疗方法。囊性纤维化（CF）是一种遗传性疾病，在美国每2500名出生的儿童中就有1人受到影响。这种疾病影响呼吸和消化，目前还没有治愈的方法。CF患者不能将盐（氯化钠）移进或移出某些细胞，包括肺和胰腺的细胞，从而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，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.