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 跨膜电导调节器（CFTR）为了确定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，在那里它起作用， cAMP激活的氯离子通道CFTR介导的跨极化上皮细胞的Cl-转运受 调节通道活性和通过控制质膜中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， F508-CFTR不能有效地从内质网输出，并且因为质膜 半衰期 F508-CFTR降低。降低质膜半衰期的机制 F508-CFTR是 还没有完全理解，部分原因是促进内吞运输的蛋白质相互作用， CFTR在质膜尚未完全阐明。在初步研究中，我们发现 在人气道上皮细胞中调节CFTR在顶膜处的运输的几种蛋白质。 阐明这些蛋白在CFTR运输中的作用对于理解顶膜至关重要。 贩运缺陷 F508-CFTR。因此，我们建议检验气道细胞顶端 WT-CFTR的膜密度和 F508-CFTR受蛋白质相互作用的差异调节， 在它们从顶膜内化的过程中，它们沿着内吞途径运输，并分选为 回收或降解。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 Dab 2通过促进CFTR而抑制CFTR在顶膜中的表达的假说 气道上皮细胞的内吞作用。这个特定目的的目标是阐明Dab 2在CFTR中的作用 内吞作用，并检查是否 F508突变加速Dab 2介导的内吞作用， CFTR。具体目标#2检验c-Cbl抑制CFTR在根尖细胞中表达的假设。 通过促进气道上皮细胞中的CFTR内吞作用来调节细胞膜。这一具体目标的目的是 阐明c-Cbl及其衔接蛋白CIN 85在CFTR内吞作用中的作用，并确定c-Cbl及其衔接蛋白CIN 85在CFTR内吞作用中是否起作用。 F508突变加速c-Cbl介导的CFTR的内吞作用。具体目标#3检验这一假设 Rab 4通过分选内化的CFTR抑制CFTR在顶膜中的表达， 气道上皮细胞中的溶酶体降解。这一具体目标的目的是阐明 Rab 4在靶向内化CFTR降解中的作用，并确定Rab 4是否 F508突变加速 Rab 4介导的内化CFTR的分选。我们预期，我们的研究，在人体气道进行， 上皮细胞：（1）将扩大我们对CFTR内吞运输的理解;（2）将阐明 降低质膜半衰期的机制 F508-CFTR;和（3）将导致新的治疗 CF患者的方法。叙事 囊性纤维化（CF）是一种遗传性疾病，在美国每2,500名出生的儿童中就有一名受到影响。的 这种疾病影响呼吸和消化，目前还没有治愈这种疾病的方法。CF患者不能移动 盐（氯化钠）进出某些细胞，包括那些排列在肺和胰腺上的细胞， 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 这种药物将恢复盐进出细胞的运动，减轻CF患者的症状。