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传导的基因突变引起的