CFTR Biogenesis and Function in Epithelia

描述（由申请人提供）：囊性纤维化跨膜传导调节剂（CFTR）是一种camp依赖性蛋白激酶a （PKA）激活的氯离子通道，存在于许多上皮细胞类型的顶端表面。该蛋白的一个不寻常的特征是，在生物发生过程中，多达75%的CFTR被内质网（ER）相关降解（ERAD）途径降解，这表明CFTR本质上是不稳定的。DeltaF508是CFTR中最常见的突变，由折叠缺陷引起，并被ERAD完全降解。由于在上皮细胞中合成的CFTR数量有限，先前的CFTR成熟研究使用了异源的过表达系统。我们在气道上皮细胞系Calu-3中的初步数据表明，所有新合成的野生型CFTR都转化为该蛋白的成熟糖基化形式。这些数据质疑野生型CFTR是否不稳定，并允许我们提出以下假设：野生型CFTR内源性表达缓慢，但在上皮细胞中有效。考虑到ERAD对野生型蛋白降解的过高估计，我们想知道deltaF508是否也可能如此。此外，低温处理后，deltaF508 CFTR可以释放到表面，最近的研究报道，部分deltaF508在某些上皮表面表达。基于此，我们假设在某些上皮中，一些deltaF508逃脱了ERAD并被运送到根尖表面。为了验证这些假设，我们提出了以下具体目标：(1)确定极化上皮中野生型CFTR生物发生的效率、运输动力学和途径；(2)验证deltaF508 CFTR的成熟和传递到细胞表面存在细胞类型特异性差异的假设。为了完成这些目标，我们将在极化条件下利用气道、胰腺和结肠/肠上皮来跟踪野生型和deltaF508的成熟和表面递送。我们将使用代谢脉冲追踪实验来监测成熟度和蛋白质半衰期，使用生化和形态学方法通过分泌途径跟踪CFTR的命运，并使用敏感的表面生物素化试验和ususing chamber分析来跟踪表面稳定性和功能。这些实验的完成将为生理学上重要的多领域提供新的重要信息；整体膜蛋白是在许多上皮细胞中合成的。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A (PKA)-activated chloride channel that is found on the apical surface of a number of cell types of epithelia. One unusual feature of this protein is that during biogenesis, as much as 75% of CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway, suggesting that CFTR is intrinsically unstable. DeltaF508, the most common mutation in CFTR, results from a folding defect and is completely degraded by ERAD. Previous studies on CFTR maturation utilized heterologous, over-expression systems because of the limited amounts of CFTR that are synthesized in epithelia. Our preliminary data in an airway epithelial cell line, Calu-3, suggest that all of the newly synthesized wild-type CFTR is converted to the maturely glycosylated form of this protein. These data question whether wild-type CFTR is unstable and allow us to pose the following hypothesis: endogenous expression of wild-type CFTR is slow, but efficient in epithelial cells. Given the over-estimation of the degradation of the wild-type protein by ERAD, we wonder if this might also be true for the deltaF508. Additionally, deltaF508 CFTR can be released to the surface after low temperature treatment, and recent studies report that some deltaF508 is expressed on the surface of certain epithelia. Based on this, we hypothesize that in certain epithelia some deltaF508 escapes ERAD and is delivered to the apical surface. To test these hypotheses, we propose the following specific aims: (1) to determine the efficiency, transport kinetics, and pathway of wild-type CFTR biogenesis in polarized epithelia; and (2) to test the hypothesis that there are cell-type specific differences in deltaF508 CFTR maturation and delivery to the cell surface. To complete these aims, we will utilize airway, pancreatic, and colonic/intestinal epithelia under polarized conditions to follow wild type and deltaF508 maturation and surface delivery. We will monitor maturation and protein half-life using metabolic pulse-chase experiments, follow the fate of CFTR through the secretory pathway using both biochemical and morphological approaches, and follow the surface stability and function using a sensitive surface biotinylation assay and Ussing chamber analysis. Completion of these experiments will provide novel and important information on a physiologically important, multi-domain; integral membrane protein is synthesized in a number of epithelial cell types.