Cell Biology of CFTR in Polarized Epithelia

超出所提供的空间。囊性纤维化（CF）是由编码囊性纤维化跨膜传导调节因子（CFTR）的基因突变引起的，CFTR是一种在上皮细胞中发现的氯离子通道。我们实验室之前的研究表明，CFTR经历快速内吞作用，这表明这提供了一种调节CFTR表面表达的机制。在CF中，超过70%的患者有一个特定的突变，AF508，导致产生错误折叠的蛋白质，无法退出内质网（ER）。因此，由于AF508具有生物活性，一种明显的治疗方法是开发将AF508 CFTR释放到细胞表面的方法。然而，最近的研究表明，与野生型蛋白不同，AF508 CFTR经过化学伴侣或低温处理后迅速从表面去除并降解，到达细胞表面。由于野生型CFTR表面表达调控机制尚不清楚，因此AF508 CFTR表面稳定性缺陷尚不清楚。我们的假设是，野生型CFTR通过与上皮特异性蛋白（如EBP50）的相互作用在细胞表面稳定下来，由于其细胞质尾部区域的分选信号，通过网格蛋白包被的凹孔被内化，并通过囊泡运输有效地循环回到细胞表面。此外，我们假设CFTR的磷酸化状态调节这些过程，更重要的是，CFTR内的突变显著影响CFTR在细胞表面的运输和稳定性。为了验证这些假设，我们将(1)定义野生型CFTR在气道上皮细胞中的运输，(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CFTR细胞生物学的三个关键方面：(1)质膜停留时间；(2)胞吞作用的细胞机制；(3) CFTR循环利用程度。使用简化的人气道上皮细胞CFTR生物素化内化试验，我们将评估野生型CFTR与CFTR突变体（包括AF508、R31L、Y1424A/I1427A和ATRL）的CFTR运输的细胞生物学和生理学特性。了解CFTR通常存在的上皮细胞中CFTR表面表达、内化和稳定性的正常动态将为CFTR的基本细胞生物学提供有价值的信息，并将为CF中最常见的突变提供潜在的治疗方法。PERFORMANCE SITE ========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================