Cell Biology of CFTR in Polarized Epithelia

囊性纤维化（CF）是由编码囊性纤维化跨膜传导的基因突变引起的 调节因子（CFTR），一种在上皮细胞中发现的氯离子通道。我们实验室以前的研究 证明CFTR经历快速的内吞作用，这表明这提供了一种调节CFTR的机制。 CFTR表面表达。在CF中，超过70%的患者具有一种特异性突变AF 508，其导致 不能离开内质网的错误折叠蛋白质的产生。明显的治疗 因此，方法是开发将AF 508 CFTR释放到细胞表面的方法，因为AF 508具有 生物活性然而，最近的研究表明，AF 508 CFTR到达细胞表面， 在化学伴侣或低温处理后， 野生型蛋白质。由于对野生型CFTR表面表达如何调节知之甚少， AF 508 CFTR表面稳定性的缺陷尚不清楚。我们的假设是野生型CFTR在细胞中是稳定的， 通过与上皮特异性蛋白如EBP 50的相互作用，表面通过网格蛋白内化， 由于在其细胞质尾区分选信号，因此被涂覆的凹坑，并被有效地回收回细胞 表面通过囊泡运输。此外，我们假设CFTR的磷酸化状态调节这些变化。 更重要的是，CFTR内的突变显著影响CFTR的运输和稳定性 在细胞表面。为了验证这些假设，我们将（1）定义气道上皮细胞中野生型CFTR的运输， 细胞和（2）定义CFTR突变如何影响内吞作用和再循环。我们将重点关注三个关键 CFTR细胞生物学方面：（1）质膜停留时间;（2）内吞作用的细胞机制; （3）CFTR的再循环程度。使用人CFTR的简化生物素化内化测定 我们将评估呼吸道上皮细胞CFTR运输的细胞生物学和生理学特性， 野生型CFTR与包括AF 508、R31 L、Y1424 A/I1427 A和ATRL的一组选定CFTR突变体的比较。 了解CFTR表面表达的正常动力学，内在化和上皮细胞的稳定性 CFTR通常驻留的位置将提供有关细胞生物学基础的有价值的信息， CFTR，并将为CF中最常见的突变提供潜在的治疗方法。

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.