Regulation of the Endocytic Trafficking of CFTR

我们的长期目标是阐明囊性纤维化的细胞内转运途径 跨膜电导调节器(CFTR)为了识别该致命疾病的治疗靶点， 囊性纤维化(CF)。CFTR在上皮细胞的顶端质膜中表达，在那里它的功能是 CAMP激活的氯离子通道。CFTR介导的氯离子跨极化上皮细胞的转运受 通过调节通道活性和控制质膜中CFTR通道的数量。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输出，因为质膜 降低了F508-CFTR的半衰期。F508-CFTR减少质膜半衰期的机制是 尚未完全了解，部分原因是促进细胞内转运的蛋白质相互作用 细胞膜上的CFTR还没有完全阐明。在初步研究中，我们发现 调节人呼吸道上皮细胞顶膜CFTR转运的几种蛋白质。 阐明这些蛋白在cftr运输中的作用对于理解根尖膜至关重要。 F508-CFTR的贩运缺陷。因此，我们建议检验这样一种假设，即呼吸道细胞顶端 WT-CFTR和F508-CFTR的膜密度受发生的蛋白质相互作用的差异调节 在它们从顶膜内化的过程中，沿着内吞途径运输，并对 要么回收利用，要么降解。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 DAB2通过促进CFTR而抑制CFTR在根尖膜中表达的假说 呼吸道上皮细胞的内吞作用。这一特定目的的目的是阐明DAB2在CFTR中的作用 并检测F508突变是否加速DAB2介导的细胞内吞作用。 CFTR。特殊目的#2.检验c-Cb1抑制CFTR在根尖的表达的假设 通过促进呼吸道上皮细胞内吞CFTR膜。这一具体目标的目标是 阐明c-Cbl及其接头蛋白CIN85在CFTR内吞作用中的作用，并确定c-Cbl及其接头蛋白CIN85 F508突变可加速c-Cbl介导的CFTR内吞作用。具体目标#3.检验假设 Rab4通过分选内化的CFTR抑制CFTR在根尖膜中的表达 呼吸道上皮细胞中溶酶体的降解。这一特定目标的目的是阐明 Rab4靶向内化的CFTR进行降解并确定F508突变是否加速 Rab4介导内化CFTR的分选。我们预计，我们在人体呼吸道中进行的研究 上皮细胞：(1)将扩大我们对CFTR内吞运输的理解；(2)将阐明 F508-CFTR减少质膜半衰期的机制；以及(3)将导致新的治疗方法 纤维支气管炎患者的治疗方法。叙述性 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。这个 这种疾病会影响呼吸和消化，目前还没有治愈的方法。Cf患者不能移动 盐(氯化钠)流入和流出某些细胞，包括那些排列在肺和胰腺内的细胞以及作为 结果产生粘稠的粘液和其他分泌物。这个应用程序的长期目标是开发一个 一种可以恢复盐分进出细胞并缓解CF患者症状的药物。

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. Narrative 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.