Cell Biology of CFTR in Polarized Epithelia

极化上皮细胞 CFTR 的细胞生物学

• 批准号: 7154139
• 负责人: JAMES F. COLLAWN
• 金额: $ 27.5万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-20 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7154139
• 关键词: Cell; Biology; CFTR; Polarized; Epithelia

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel found in epithelial cells. Previous studies from our laboratory have demonstrated that CFTR undergoes rapid endocytosis, suggesting that this provides a mechanism for regulating CFTR surface expression. In CF, more than 70% of patients have one specific mutation, AF508, which results in the production of a misfolded protein that fails to exit the endoplasmic reticulum (ER). An obvious therapeutic approach, therefore, is to develop methods for releasing AF508 CFTR to the cell surface, since AF508 has biological activity. Recent studies, however, have demonstrated the AF508 CFTR that reaches the cell surface after chemical chaperone or low temperature treatment is rapidly removed from the surface and degraded, unlike the wild type protein. Since little is known about how wild type CFTR surface expression is regulated, the defect in AF508 CFTR surface stability is unclear. Our hypothesis is that wild type CFTR is stabilized at the cell surface through interactions with epithelial-specific proteins such as EBP50, is internalized through clathrin- coated pits because of sorting signals in its cytoplasmic tail regions, and is efficiently recycled back to the cell surface via vesicle trafficking. Further, we hypothesize that the phosphorylation status of CFTR regulates these processes and more importantly, mutations within CFTR dramatically influence CFTR trafficking and stability at the cell surface. To test these hypotheses, we will (1) define wild type CFTR trafficking in airway epithelial cells and (2) define how mutations in CFTR affect endocytosis and recycling. We will focus on three key aspects of CFTR cell biology: (1) plasma membrane residence time; (2) cellular mechanisms of endocytosis; and (3) degree of CFTR recycling. Using simplified biotinylation internalization assays of CFTR in human airway epithelial cells, we will assess the cell biological and physiological properties of CFTR trafficking of wild type CFTR versus a select panel of CFTR mutants that include AF508, R31L, Y1424A/I1427A, and ATRL. Understanding the normal dynamics of CFTR surface expression, internalization, and stability in epithelial cells where CFTR normally resides will provide valuable information regarding the fundamental cell biology of CFTR and will provide potential therapies for the most common mutation in CF.

囊性纤维化（CF）是由编码囊性纤维化跨膜电导的基因突变引起的 调节剂（CFTR），一种在上皮细胞中发现的氯化物通道。我们实验室的先前研究 证明CFTR经历了快速的内吞作用，这表明这提供了调节的机制 CFTR表面表达。在CF中，超过70％的患者具有一个特定的突变AF508，这导致 未能退出内质网（ER）的错误折叠蛋白的产生。一个明显的治疗性 因此，方法是开发用于将AF508 CFTR释放到细胞表面的方法，因为AF508具有 生物活性。然而，最近的研究证明了到达细胞表面的AF508 CFTR 化学伴侣或低温处理后，从表面迅速去除并降解，与 野生型蛋白质。由于对野生型CFTR表面表达的调节知之甚少，因此 AF508 CFTR表面稳定性中的缺陷尚不清楚。我们的假设是野生型CFTR在细胞上稳定 通过与上皮特异性蛋白（例如EBP50）相互作用的表面通过网格蛋白 - 涂层凹坑是因为在其细胞质尾部区域排序信号，并有效地回收回细胞 通过囊泡运输表面。此外，我们假设CFTR的磷酸化状态调节这些状态 过程，更重要的是，CFTR内的突变极大地影响CFTR运输和稳定性 在细胞表面。为了检验这些假设，我们将（1）定义气道上皮中的野生型CFTR贩运 细胞和（2）定义CFTR突变如何影响内吞和回收。我们将专注于三个钥匙 CFTR细胞生物学的各个方面：（1）质膜停留时间； （2）内吞作用的细胞机制； （3）CFTR回收程度。使用人类中CFTR的简化生物素化测定法 气道上皮细胞，我们将评估CFTR运输的细胞生物学和生理特性 野生型CFTR与包括AF508，R31L，Y1424A/I1427A和ATRL的CFTR突变体的精选面板。 了解上皮细胞中CFTR表面表达，内在化和稳定性的正常动力学 CFTR通常居住的地方将提供有关基本细胞生物学的有价值信息 CFTR并将为CF中最常见的突变提供潜在的疗法。