Denning G M, Ostedgaard L S, Cheng S H, Smith A E, Welsh M J
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
J Clin Invest. 1992 Jan;89(1):339-49. doi: 10.1172/JCI115582.
Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.
囊性纤维化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。为了进一步了解CFTR的功能和调节机制，我们使用共聚焦免疫荧光显微镜将CFTR定位在经针对该蛋白不同区域的单克隆抗体染色的细胞中：R（调节）结构域（M13 - 1）、COOH末端（M1 - 4）以及一个预测的细胞外结构域（M6 - 4）。所有这三种抗体都能从表达CFTR的细胞中免疫沉淀出一条155 - 170 kD的多肽。每种抗体都能对表达重组CFTR的HeLa细胞和3T3细胞进行染色，但不能对缺乏内源性CFTR的细胞进行染色：HeLa细胞、NIH - 3T3细胞和内皮细胞。对于定位研究，我们使用了表达内源性CFTR且具有cAMP激活的顶端Cl⁻通透性的上皮细胞系：T84细胞、CaCo2细胞和HT29克隆19A细胞。我们的结果表明，CFTR在这些上皮细胞中是一种顶端膜蛋白，原因如下：（a）CFTR的染色类似于几种顶端膜标记物的染色，但与基底外侧膜蛋白的染色不同；（b）细胞单层的薄片显示顶端膜有染色；（c）细胞外结构域抗体M6 - 4对未通透的细胞的顶端表面进行了染色。我们的结果并不排除CFTR也位于顶端膜下方的可能性。细胞内cAMP水平的升高并没有改变CFTR在顶端膜的染色模式。此外，cAMP介导的顶端膜Cl⁻电导增加并不需要通过囊泡与顶端膜融合来插入通道。这些结果表明，CFTR位于Cl⁻分泌上皮细胞的顶端质膜中，这一结果与Cl⁻TR是一种顶端膜氯通道的结论一致。
