Regulation of the Endocytic Trafficking of CFTR

CFTR 内吞贩运的监管

• 批准号: 7842159
• 负责人: Agnieszka Swiatecka-Urban
• 金额: $ 29.02万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842159
• 关键词: Adaptor Signaling Protein; Affect; Alzheimer' s Disease; Apical; Breathing; Cell Line; Cell membrane; Cell model; Cells; Child; Clathrin; Complex; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Digestion; Disease; Endocytosis; Endoplasmic Reticulum; Epithelial Cells; Familial Hypercholesterolemia; Goals; Half-Life; Human; Huntington Disease; Individual; Inherited; Interstitial Lung Diseases; Lead; Lung; Mediating; Membrane Protein Traffic; Molecular; Movement; Mucous body substance; Mutation; Pancreas; Pathway interactions; Pharmaceutical Preparations; Protein C Inhibitor; Protein Dynamics; Proteins; Publishing; Recruitment Activity; Recycling; Regulation; Role; Sodium Chloride; Sorting - Cell Movement; Symptoms; Testing; Thick; Tissues; apical membrane; cystic fibrosis patients; density; effective therapy; improved; intermolecular interaction; novel therapeutic intervention; protein transport; proto-oncogene protein c-cbl; research study; respiratory; therapeutic target; trafficking; ubiquitin ligase

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.

我们的长期目标是阐明囊性纤维化的内吞运输途径 跨膜电导调节剂（CFTR）为了确定致命疾病的治疗靶标， 囊性纤维化（CF）。 CFTR在上皮细胞的顶端质膜中表达，其作用为 一个营地激活的Cl-通道。 CFTR介导的Cl-跨两极化上皮细胞的转运受 通过控制质膜中的CFTR通道的数量来调节通道活动。 F508， CF中最常见的突变会减少质膜中CFTR通道的数量，因为 F508-CFTR并未有效从内质网出口，因为质膜 F508-CFTR的半衰期减少了。 F508-CFTR的质膜半衰期降低的机制为 一定程度地不完全理解，因为 质膜处的CFTR尚未完全阐明。在初步研究中，我们确定了 几种调节CFTR在人类气道上皮细胞中的顶端膜上运输的蛋白质。 阐明这些蛋白质在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，我们建议测试气道细胞顶端的假设 WT-CFTR和F508-CFTR的膜密度受到发生的蛋白质相互作用的调节 在其内在化中，根尖膜，沿着内吞途径进行贩运，并分类 回收或退化。为了检验这一假设，我们提出了三个特定目的：特定目的＃1。测试 DAB2通过促进CFTR抑制CFTR在顶端膜中CFTR的表达的假设 气道上皮细胞中的内吞作用。该特定目的的目的是阐明DAB2在CFTR中的作用 内吞作用并检查F508突变是否加速了DAB2介导的内吞作用 CFTR。特定目标＃2。检验C-CBL抑制CFTR在顶端的表达的假设 通过促进气道上皮细胞中CFTR内吞作用的膜。这个特定目的的目的是 阐明C-CBL及其衔接蛋白的作用CIN85在CFTR内吞作用中，并确定是否是否 F508突变加速了C-CBL介导的CFTR内吞作用。特定目标＃3。检验假设 RAB4通过对内部化CFTR排序的CFTR来抑制CFTR在顶膜中的表达 气道上皮细胞中的溶酶体降解。这个特定目的的目的是阐明 Rab4靶向内部CFTR进行降解，并确定F508突变是否加速 RAB4介导的内在CFTR的排序。我们预计我们的研究在人类气道中进行 上皮细胞：（1）将扩展我们对CFTR内吞交易的理解； （2）将阐明 F508-CFTR的质膜半衰期降低的机制； （3）将导致新的治疗 CF患者的方法。叙述 囊性纤维化（CF）是一种遗传性疾病，每2500名在美国出生的儿童中有一个。这 疾病会影响呼吸和消化，目前无法治愈该疾病。 CF患者无法移动 盐（氯化钠）进入某些细胞，包括肺和胰腺排成一列的细胞 结果会产生浓稠的粘性粘液和其他分泌物。该应用程序的长期目标是开发 将恢复盐进出细胞的药物，并减轻CF患者的症状。