Regulation of the Endocytic Trafficking of CFTR

描述(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)的细胞内转运途径，以确定囊性纤维化(CF)这一致命疾病的治疗靶点。CFTR在上皮细胞的顶端质膜中表达，在那里它作为cAMP激活的氯通道发挥作用。Cftr介导的氯离子跨极化上皮细胞的转运是通过调节通道活性和控制质膜上cftr通道的数量来调节的。F508是CF中最常见的突变，由于F508-CFTR不能有效地从内质网输出，以及F508-CFTR的质膜半衰期缩短，导致质膜上CFTR通道的数量减少。F508-CFTR减少质膜半衰期的机制还不完全清楚，部分原因是促进CFTR在质膜上内吞转运的蛋白质相互作用尚未完全阐明。在初步研究中，我们鉴定了几种调节CFTR在人呼吸道上皮细胞顶膜运输的蛋白质。阐明这些蛋白在CFTR转运中的作用对于理解F508-CFTR的根尖膜转运缺陷至关重要。因此，我们建议检验这样一种假设，即WT-CFTR和？F508-CFTR在从顶膜内化、沿内吞途径运输以及回收或降解过程中发生的蛋白质相互作用对呼吸道细胞顶膜密度有不同的调节作用。为了验证这一假设，我们提出了三个特定目标：特定目标#1.验证DAB2通过促进呼吸道上皮细胞CFTR内吞来抑制CFTR在顶膜的表达的假设。本研究的目的是阐明DAB2在CFTR内吞作用中的作用，并研究F508突变是否加速了DAB2介导的CFTR内吞作用。特殊目的#2.验证c-Cbl通过促进呼吸道上皮细胞CFTR内吞来抑制CFTR在根尖膜上的表达的假设。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突变是否加速了c-Cbl介导的CFTR内吞作用。具体目的#3.通过对呼吸道上皮细胞溶酶体降解的内化CFTR进行分选，验证Rab4抑制CFTR在根尖膜上表达的假设。本研究的目的是阐明Rab4在靶向内化CFTR降解中的作用，并确定F508突变是否加速了Rab4介导的内化CFTR的分选。我们预计，我们在人类呼吸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)将阐明F508-CFTR质膜半衰期减少的机制；(3)将为CFTR患者提供一种新的治疗方法。与公共卫生相关。囊性纤维化是一种遗传性疾病，在美国每2500名出生的儿童中就有一名受到影响。这种疾病会影响呼吸和消化，目前还没有治愈方法。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内的细胞，从而产生粘稠的粘液和其他分泌物。这一应用的长期目标是开发一种药物，可以恢复盐在细胞内和细胞外的移动，并缓解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.