A PDZ-interacting domain in CFTR is an apical membrane polarization signal
Bryan D. Moyer, … , Min Li, Bruce A. Stanton
Bryan D. Moyer, … , Min Li, Bruce A. Stanton
Published November 15, 1999
Citation Information: J Clin Invest. 1999;104(10):1353-1361. https://doi.org/10.1172/JCI7453.
View: Text | PDF
Article Article has an altmetric score of 3

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Abstract

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

Authors

Bryan D. Moyer, Jerod Denton, Katherine H. Karlson, Donna Reynolds, Shusheng Wang, John E. Mickle, Michal Milewski, Garry R. Cutting, William B. Guggino, Min Li, Bruce A. Stanton

×

Figure 1

Options: View larger image (or click on image) Download as PowerPoint
The COOH-terminal PDZ-interacting domain (TRL) is required for polarizat...
The COOH-terminal PDZ-interacting domain (TRL) is required for polarization of CFTR to the apical membrane. (a) Confocal fluorescence micrograph (xz plane) of MDCK cells stably expressing GFP-wt-CFTR in the apical membrane. (b) Confocal fluorescence micrograph (xz plane) of MDCK cells stably expressing GFP-CFTR-ΔTRL in the apical and lateral membranes. GFP fluorescence is green, and ZO-1, a protein in tight junctions that separates apical and basolateral membrane domains, is red. Scale bar = 10 μm. AP = location of apical membrane; BL = location of basal membrane. (c) Western blot of cells expressing wt-CFTR or CFTR-ΔTRL. Selective cell-surface biotinylation of the apical (AP) or basolateral membrane (BL). Whereas GFP-wt-CFTR was expressed primarily in the apical membrane (ratio of apical/basolateral membrane expression = 7.5), GFP-CFTR-ΔTRL was expressed nearly equally in the apical and basolateral membranes (ratio of apical/basolateral membrane expression = 0.6). Mature, glycosylated GFP-wt-CFTR band C is approximately 240 kDa. *High molecular weight form of CFTR that has been reported previously (33, 37). (d) Selective biotinylation of the apical membrane. (e) Selective biotinylation of the basolateral membrane. Biotin was detected with streptavidin-Texas-red as described (33). Images in d and e demonstrate that in our experiments, tight junctions were intact and the biotin regent applied to the apical membrane did not have access to CFTR present on the basolateral membrane and vice versa. In addition, the absence of core-glycosylated GFP-wt-CFTR (band B) in surface biotinylated samples indicates that cell integrity was not compromised and that biotin was not accessible to the endoplasmic reticulum and cis-Golgi apparatus, where core glycosylated CFTR band B is located.