ABCMdb

PMID: 12509457

Estell K, Braunstein G, Tucker T, Varga K, Collawn JF, Schwiebert LM
Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif.
Mol Cell Biol. 2003 Jan;23(2):594-606., [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCC7 p.Trp1282* Experiments employed IB3-1 cells (bronchial; ⌬F508/W1282X compound heterozygote; a gift from Pam Zeitlin, Johns Hopkins University, Baltimore, Md.) (44). Login to comment 57 ABCC7 p.Gly551Asp The CFTR-encoding constructs utilized were pRSV-WT-CFTR (9) (a gift from Erik M. Schwiebert, University of Alabama at Birmingham), pGFP-WT-CFTR, pGFP-⌬F508, pGFP-G551D, and pGFP-⌬TRL (18) (all pGFP constructs were generous gifts from Bruce A. Stanton, Dartmouth Medical School). Login to comment 110 ABCC7 p.Trp1282* For these experiments, cells of IB3-1, a CF airway epithelial cell line that is heterozygous for the CFTR mutation (W1282X/ ⌬F508 [44]), were utilized. Login to comment 147 ABCC7 p.Gly551Asp For these experiments, we took advantage of the ⌬F508, G551D, and ⌬TRL CFTR mutations, which differ in their levels of chloride transport activity. Login to comment 165 ABCC7 p.Gly551Asp In contrast, G551D is a missense mutation within NBD-1 resulting in the conversion of a glycine residue to an aspartic acid residue. Login to comment 166 ABCC7 p.Gly551Asp Unlike ⌬F508, the G551D mutation traffics normally to the plasma membrane; however, it is unable to function as a chloride channel (43). Login to comment 169 ABCC7 p.Gly551Asp In these experiments, IB3-1 cells were transfected with constructs encoding WT-CFTR, ⌬F508-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control. Login to comment 173 ABCC7 p.Gly551Asp In sharp contrast, cells expressing ⌬F508-CFTR, G551D-CFTR, or the mock control were not responsive to the effects of cAMP agonists. Login to comment 176 ABCC7 p.Gly551Asp In sharp contrast, G551D restored RANTES expression to a level slightly less than but comparable to that observed with WT-CFTR (Fig. 5). Login to comment 182 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As shown in Fig. 6, biotinylation analysis revealed that WT-CFTR, G551D-CFTR, and ⌬TRL-CFTR were each expressed at the cell surface; WTand ⌬TRL-CFTR surface expression levels were comparable while G551D-CFTR expression was detectable yet significantly less than that of the others. Login to comment 184 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because WTand G551D-CFTR, but neither ⌬TRL- nor ⌬F508-CFTR, were able to restore RANTES expression, these results suggest that insertion of an intact CFTR molecule into the plasma membrane is necessary for CFTR-dependent RANTES expression. Login to comment 190 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp IB3-1 cells were cotransfected with constructs encoding beta-galactosidase (pSV-beta-galactosidase; 0.5 ␮g/well) and mock control (pEGFP-C1; open circles) or WT-CFTR (pGFP-WT-CFTR; closed circles) (A) or G551D-CFTR (pGFP-G551D; gray circles), ⌬F508-CFTR (pGFP-⌬F508; open circles), or ⌬TRL-CFTR (pGFP-⌬TRL; closed circles) (B) (each at 1.0 ␮g/well). Login to comment 216 ABCC7 p.Gly551Asp For these experiments, IB3-1 cells were transfected with constructs encoding WT-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control and then cultured in the presence and absence of TNF-␣ and IFN-␥. Login to comment 220 ABCC7 p.Gly551Asp IB3-1 cells were cotransfected with constructs encoding beta-galactosidase and mock control, WT-CFTR, G551D-CFTR, ⌬F508-CFTR, or ⌬TRL-CFTR as described in the Fig. 4 legend and then cultured with or without TNF-␣ and IFN-␥ as described in the Fig. 1 legend. Login to comment 226 ABCC7 p.Gly551Asp As shown in Fig. 9C, cells transfected with constructs encoding G551D-CFTR displayed a pattern of enhanced NF-␬B binding that resembled the pattern of NF-␬B binding observed for cells transfected with constructs encoding WT-CFTR; in contrast, ⌬TRL-CFTR did not promote the binding of NF-␬B either in the presence or in the absence of TNF-␣ and IFN-␥ (Fig. 9C). Login to comment 229 ABCC7 p.Gly551Asp IB3-1 cells were cotransfected with constructs encoding beta-galactosidase and mock control (M) or WT-CFTR (A), G551D-CFTR or ⌬TRL-CFTR (B), or ⌬F508-CFTR (C) as described in the Fig. 4 legend. Login to comment 270 ABCC7 p.Gly551Asp (C) IB3-1 cells were transfected with constructs encoding beta-galactosidase, WT-CFTR, G551D-CFTR, or ⌬TRL-CFTR; stimulated for 30 min with and without TNF-␣-IFN-␥ (denoted by ϩ and -, respectively); and then prepared for EMSA. Login to comment 281 ABCC7 p.Gly551Asp For these experiments, IB3-1 cells were transfected transiently with constructs encoding WT-CFTR, G551D-CFTR, ⌬TRL-CFTR, or a mock control; cultured in the presence and absence of TNF-␣ and IFN-␥; and then prepared for EMSA. Login to comment 285 ABCC7 p.Gly551Asp As shown in Fig. 11C, cells transfected with constructs encoding G551D-CFTR displayed a pattern of enhanced NF-␬B binding that resembled the pattern of NF-␬B binding observed for cells transfected with constructs encoding WT-CFTR; in contrast, ⌬TRL-CFTR did not promote the binding of NF-␬B either in the presence or in the absence of TNF-␣ and IFN-␥ (Fig. 11C). Login to comment 297 ABCC7 p.Gly551Asp Analyses of the CFTR mutants G551D- and ⌬TRL-CFTR, which differ in their ability to transport chloride and restore RANTES expression, support these observations. Login to comment 300 ABCC7 p.Gly551Asp Data obtained through the use of CFTR disease-associated (G551D and ⌬F508) and truncation (⌬TRL) mutations presented herein FIG. 11. Login to comment 306 ABCC7 p.Gly551Asp (C) IB3-1 cells were transfected with constructs encoding beta-galactosidase, WT-CFTR, G551D-CFTR, or ⌬TRL-CFTR; stimulated with and without TNF-␣-IFN-␥ (denoted by ϩ and -, respectively); and then prepared for EMSA as described in the Fig. 9 legend. Login to comment 310 ABCC7 p.Gly551Asp First, the ability of G551D, but not ⌬F508, to restore RANTES expression to a level comparable with that observed with WT-CFTR suggests that CFTR molecules inserted into the plasma membrane restore RANTES expression. Login to comment 314 ABCC7 p.Gly551Asp It should be noted that, although ⌬F508- and G551D-CFTR differ in their ability to restore RANTES expression, both of these CFTR mutations are associated with severe forms of CF. Login to comment 315 ABCC7 p.Gly551Asp If the lack of RANTES expression is an important contributor to CF-associated airway inflammation, then it would be expected that neither ⌬F508 nor G551D would support RANTES expression; however, this was not the response that was observed. Login to comment 316 ABCC7 p.Gly551Asp With regard to the correlation of ⌬F508- and G551D-CFTR mutations, RANTES expression, and severe CF pulmonary disease, we note that Mickle and Cutting have reported that, despite its being a monogenic disorder, the genotype-phenotype relationship in CF is complex (14). Login to comment 321 ABCC7 p.Gly551Asp Specifically, reports by Prince and coworkers indicate that cells expressing the CFTR mutation ⌬F508 or G551D as well as non-CFTR-expressing cells display increased NF-␬B activation in response to a cytokine or P. aeruginosa stimulus compared with non-CF and "CFTR-corrected" cells; assessments were determined via measurements of NF-␬B binding, NF-␬B reporter construct activity, and NF-␬B nuclear translocation (42). Login to comment 326 ABCC7 p.Gly551Asp In contrast, results presented herein suggest that human airway epithelial cells expressing WT-CFTR or G551D-CFTR, but not ⌬TRL-CFTR, exhibit enhanced NF-␬B activation in the presence of TNF-␣ and IFN-␥ compared with mock controls. Login to comment