Regulation of the Endocytic Trafficking of CFTR

CFTR 内吞贩运的监管

• 批准号: 7652301
• 负责人: Agnieszka Swiatecka-Urban
• 金额: $ 38.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-08 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652301
• 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; public health relevance; research study; respiratory; therapeutic target; trafficking; ubiquitin ligase

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.

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电导调节剂（CFTR）的内吞运输途径，以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，在该细胞中，它充当了cAMP激活的Cl-通道。 CFTR介导的跨两极分化上皮细胞的CL传输通过调节通道活性和控制质膜中CFTR通道的数量来调节。 CF中最常见的突变是质膜中CFTR通道数量的数量，因为？F508-CFTR的数量没有有效地从内质网中导出，并且由于质膜膜的半衰期是？F508-CFTR的质膜半衰期。尚未完全理解降低？F508-CFTR的质膜半衰期的机制，部分原因是，促进CFTR在质膜上促进CFTR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，这些蛋白质调节了人类气道上皮细胞中的CFTR的运输。阐明这些蛋白质在CFTR运输中的作用对于理解？F508-CFTR的根尖膜运输缺陷至关重要。因此，我们建议测试以下假设：WT-CFTR和？F508-CFTR的气道细胞顶膜密度受到蛋白质相互作用的差异调节，这些蛋白质相互作用在其内部化过程中从根尖膜内部化，沿着内吞途径进行运输，并分类用于回收或降解。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通过促进气道上皮细胞中CFTR内吞作用来抑制CFTR在顶端膜中CFTR的表达的假设。该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突变是否会加速DAB2介导的CFTR内吞作用。特定目标＃2。测试C-CBL通过促进气道上皮细胞中CFTR内吞作用来抑制CFTR在顶端膜中CFTR的表达的假设。该特定目的的目的是阐明C-CBL及其衔接蛋白CIN85在CFTR内吞作用中的作用，并确定？F508突变是否会加速C-CBL介导的CFTR内吞作用。特定目标＃3。测试RAB4通过对内部化CFTR排序以使气道上皮细胞中的溶酶体降解来抑制CFTR的表达的假设。该特定目的的目的是阐明RAB4在靶向内部化CFTR进行降解中的作用，并确定？F508突变是否会加速RAB4介导的内部化CFTR分选。我们预计我们的研究在人类气道上皮细胞中进行：（1）将扩展我们对CFTR内吞交易的理解； （2）将阐明质膜半衰期降低的机制； F508-CFTR； （3）将导致CF患者的新治疗方法。公共卫生相关性。囊性纤维化（CF）是一种遗传性疾病，每2500名在美国出生的儿童中有一个。该疾病会影响呼吸和消化，目前无法治愈该疾病。 CF患者无法将盐（氯化钠）进入某些细胞，包括肺部和胰腺排列的细胞，结果会产生浓稠的粘粘液和其他分泌物。该应用的长期目标是开发一种可以恢复盐运动进出细胞的药物，并减轻CF患者的症状。