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.

Deletion of the PDZ-interacting domain of CFTR abrogates colocalization of CFTR and EBP50. Confocal fluorescence micrographs (xz plane) of MDCK cells coexpressing: (a–c) EBP50 and wt-CFTR, (d–e) EBP50 and CFTR-ΔTRL, and (g–i) EBP50 and CFTR-S1455X. GFP-CFTR is green (a, d, g), EBP50 is red (b, e, h), and the merged red and green images are shown in c, f, and i. Colocalization of EBP50 and GFP-CFTR is orange-yellow in c and f. Note that some GFP-CFTR-S1455X and GFP-CFTR-ΔTRL are expressed in an intracellular compartment (d and g). Also note that only some cells express EBP50 because EBP50 is transiently expressed in approximately 10% of cells. Scale bar = 10 μm. AP = location of apical membrane; BL = location of basal membrane.

Confocal fluorescence micrographs (xz plane) of cells expressing GFP-wt-CFTR or GFP-CFTR-S1455X. GFP fluorescence is green, and ZO-1, a protein in tight junctions that separates apical and basolateral membrane domains, is red. (a) GFP-wt-CFTR is located in the apical membrane of MDCK cells. (b) GFP-CFTR-S1455X is located in the lateral membrane of MDCK cells. (c) GFP-CFTR-S1455X is located in the lateral membrane of 16HBE14o- cells. (d) GFP-CFTR-S1455X (left panel in green) colocalizes with Na⁺-K⁺-ATPase (middle panel in red) in the lateral membrane (right panel is a merge of red and green channels, yellow-orange indicates colocalization). GFP-wt-CFTR is expressed in the apical membrane of 16HBE14o- cells (image not shown). Note that some GFP-CFTR-S1455X is expressed in an intracellular compartment. Scale bars = 10 μm. AP = location of apical membrane; BL = location of basal membrane.

Coimmunoprecipitation of EBP50 and CFTR. EBP50 was coexpressed with GFP-wt-CFTR or GFP-CFTR-ΔTRL in COS-7 cells. Immunoprecipitation was conducted with a polyclonal GFP antibody (to immunoprecipitate CFTR) or a nonspecific, control IgG antibody and blots were probed with monoclonal GFP (to detect CFTR) or an anti-HA antibody to detect HA-tagged EBP50, as indicated. Cell lysates were probed for the expression of CFTR and EBP50. Top 2 blots show immunoprecipitations. Bottom 2 blots show cell lysates. Neither EBP50 nor CFTR was immunoprecipitated when a nonspecific, control IgG from nonimmune rabbit serum was used. CFTR immunoprecipitation efficiency was similar in all samples, and cell lysates expressed equivalent levels of EBP50 and CFTR fusion proteins. Thus, results cannot be attributed to differential protein expression. Similar results were obtained when EBP50 was immunoprecipitated and the blots probed with a GFP antibody to identify CFTR (data not shown). Mature glycosylated GFP-wt-CFTR band C is approximately 240 kDa. The higher molecular weight band of CFTR has been reported (33, 36).