ABCMdb

PMID: 10562297

Moyer BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA
A PDZ-interacting domain in CFTR is an apical membrane polarization signal.
J Clin Invest. 1999 Nov;104(10):1353-61., [PubMed]

Sentences

No. Mutations Sentence Comment

21 ABCC7 p.Ser1455* 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. Login to comment 35 ABCC7 p.Ser1455* Toward these ends, we made chimeric constructs in which the green fluorescent protein (GFP) was linked to either wild-type (wt) CFTR or CFTR with truncations in the COOH-terminus (i.e., CFTR-∆TRL or CFTR-S1455X) and expressed these proteins in polarized kidney epithelial cells (MDCK) and human bronchial epithelial cells (16HBE14o-). Login to comment 37 ABCC7 p.Ser1455* In addition, the CFTR mutation S1455X, which lacks the 26 COOH-terminal amino acids including the PDZ-interacting domain, is mispolarized to the lateral membrane. Login to comment 52 ABCC7 p.Ser1455* pGFP-CFTR-S1455X encodes a GFP-CFTR fusion protein lacking the 26 CFTR COOH-terminal amino acids due to mutation of codon 1455 (which encodes serine) to a premature stop codon. Login to comment 86 ABCC7 p.Ser1455* In cells expressing GFP-CFTR-S1455X, the GFP mAb recognized 2 bands with relative molecular masses of approximately 210 and 240 kDa. Login to comment 113 ABCC7 p.Ser1455* Accordingly, we examined the cellular distribution of the COOH-terminal truncation mutant, S1455X, which lacks the COOH-terminal 26 amino acids including the PDZ-interacting domain (40). Login to comment 114 ABCC7 p.Ser1455* Patients with CFTR-S1455X have defective Cl-transport in sweat ducts (40). Login to comment 115 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Examination of GFP-CFTR-S1455X localization by immunofluorescence confocal microscopy demonstrated that CFTR-S1455X was polarized to the lateral plasma 1356 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 Figure 1 The COOH-terminal PDZ-interacting domain (TRL) is required for polarization of CFTR to the apical membrane. Login to comment 130 ABCC7 p.Ser1455* GFP-CFTR-S1455X colocalized with the lateral membrane protein Na+-K+-ATPase (Figure 2d). Login to comment 131 ABCC7 p.Ser1455* Moreover, the ratio of GFP-CFTR-S1455X in the apical versus the basolateral plasma membrane was 0.2 ± 0.1 as determined by domain-selective cell-surface biotinylation (Table 1). Login to comment 133 ABCC7 p.Ser1455* That CFTR-S1455X was expressed primarily in the basolateral membrane, whereas CFTR-∆TRL was equally distributed in the apical and basolateral plasma membranes, suggests that some basolateral targeting information is suppressed or inactive in CFTR-∆TRL (see Discussion). Login to comment 136 ABCC7 p.Ser1455* ABCC7 p.Ser1455* To determine whether EPB50 interacts with and colocalizes with GFP-wt-CFTR in vivo, and whether the PDZ-interacting domain of CFTR is required for interaction and colocalization with EBP50, we coexpressed EBP50 with GFP-wt-CFTR, GFP-CFTR-∆TRL, or GFP-CFTR-S1455X in MDCK and COS cells. Examination of GFP-wt-CFTR and EBP50 by immunofluorescence confocal microscopy demonstrated that GFP-wt-CFTR and EBP50 colocalized to the apical plasma membrane of MDCK cells (Figure 3, a-c). Login to comment 139 ABCC7 p.Ser1455* ABCC7 p.Ser1455* By contrast, examination of GFP-CFTR-S1455X location by immunofluorescence confocal microscopy demonstrated that GFP-CFTR-S1455X did not colocalize with EBP50. Login to comment 140 ABCC7 p.Ser1455* GFP-CFTR-S1455X was polarized to the lateral plasma membrane, whereas EBP50 was polarized to the apical plasma membrane (Figure 3, g-i). Login to comment 141 ABCC7 p.Ser1455* To test the hypothesis that EBP50 and CFTR interact via the COOH-terminal PDZ-interacting domain of CFTR in vivo, we coexpressed EBP50 with GFP-wt-CFTR, GFP-CFTR-∆TRL, or GFP-CFTR-S1455X in COS cells and determined whether CFTR and EBP50 could be coimmunoprecipitated. Login to comment 142 ABCC7 p.Ser1455* EBP50 coimmunoprecipitated with GFP-wt-CFTR but not with GFP-CFTR-∆TRL (Figure 4) or GFP-S1455X. Login to comment 145 ABCC7 p.Ser1455* Discussion We have demonstrated 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. Our data also suggest that the polarization of CFTR to the apical plasma membrane involves interaction with an apical membrane PDZ The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1357 Table 1 Deletion of the PDZ-interacting domain of CFTR abrogates its apical polarization Cell line wt-CFTR CFTR-∆TRL CFTR-S1455X MDCK (Biotinylation) 7.5 ± 2.3 0.6A ± 0.3 0.2A ± 0.1 MDCK (Confocal) 3.0 ± 0.4 0.6A ± 0.1 0.1A ± 0.1 16HBE14o- (Confocal) 3.6 ± 0.6 0.7A ± 0.1 0.1A ± 0.1 Data are expressed as the ratio of CFTR expressed in the apical versus the basolateral plasma membrane. Login to comment 150 ABCC7 p.Ser1455* Figure 2 Confocal fluorescence micrographs (xz plane) of cells expressing GFP-wt-CFTR or GFP-CFTR-S1455X. Login to comment 153 ABCC7 p.Ser1455* (b) GFP-CFTR-S1455X is located in the lateral membrane of MDCK cells. Login to comment 154 ABCC7 p.Ser1455* (c) GFP-CFTR-S1455X is located in the lateral membrane of 16HBE14o-cells. Login to comment 155 ABCC7 p.Ser1455* (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). Login to comment 157 ABCC7 p.Ser1455* Note that some GFP-CFTR-S1455X is expressed in an intracellular compartment. Login to comment 180 ABCC7 p.Ser1455* Confocal fluorescence micrographs (xz plane) of MDCK cells coexpressing: (a-c) EBP50 and wt-CFTR, (d-e) EBP50 and CFTR-∆TRL, and (g-i) EBP50 and CFTR-S1455X. Login to comment 181 ABCC7 p.Ser1455* 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-∆TRL are expressed in an intracellular compartment (d and g). Login to comment 189 ABCC7 p.Ser1455* In the present study, we observed that approximately 30% of total cellular GFP-wt-CFTR was expressed in the plasma membrane, whereas only 1-3% of total cellular GFP-CFTR-∆TRL and GFP-S1455X was expressed in the plasma membrane. Login to comment 197 ABCC7 p.Ser1455* ABCC7 p.Ser1455* Although the polarization of CFTR-S1455X is dramatically different from wt-CFTR, this mutation does not cause CF, as pulmonary and pancreatic function appear to be normal; however, CFTR-S1455X results in elevated sweat chloride concentration (40). Login to comment 198 ABCC7 p.Ser1455* ABCC7 p.Ser1455* ABCC7 p.Ser1455* Because CFTR-S1455X is a functionally normal Cl-channel, we conclude that defective Cl-transport in sweat ducts is most likely due to the mispolarization of CFTR-S1455X to the lateral rather than the apical plasma membrane. Login to comment 199 ABCC7 p.Ser1455* We speculate that the relatively small fraction of CFTR-S1455X expressed at the apical membrane may be sufficient to sustain pulmonary and pancreatic function, but not sweat duct function. Login to comment 200 ABCC7 p.Ser1455* In addition, the fact that CFTR-S1455X was polarized to the lateral membrane, whereas CFTR-∆TRL was equally distributed between the apical and basolateral plasma membranes, suggests that some basolateral sorting information is suppressed in CFTR-∆TRL. Login to comment 202 ABCC7 p.Ser1455* Because sorting determinants are position dependent and hierarchical, deletion of the last 26 amino acids in CFTR may unmask the tyrosine and/or dileucine motifs, such that 1 or both of these motifs direct CFTR-S1455X to the lateral membrane (13, 14). Login to comment 216 ABCC7 p.Ser1455* Indeed, in MDCK cells stably expressing GFP-CFTR-∆TRL or GFP-CFTR-S1455X, we observed that CPT-cAMP-stimulated, transepithelial Cl-secretion was not different from parental, untransfected MDCK cells. Login to comment