title: Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.
authors.affiliation: Cellular Morphogenesis and Signalisation, UMR 144 CNRS-Institut Curie, Paris, France.
authors.forename: M A
authors.initials: MA
authors.lastname: Costa de Beauregard
authors.forename: A
authors.initials: A
authors.lastname: Edelman
authors.forename: D
authors.initials: D
authors.lastname: Chesnoy-Marchais
authors.forename: D
authors.initials: D
authors.lastname: Tondelier
authors.forename: A
authors.initials: A
authors.lastname: Lapillonne
authors.forename: F
authors.initials: F
authors.lastname: El Marjou
authors.forename: S
authors.initials: S
authors.lastname: Robine
authors.forename: D
authors.initials: D
authors.lastname: Louvard
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins; D009154:Mutation; D018408:Patch-Clamp Techniques; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000276:immunology / Q000378:metabolism*
chemical_list: C505032:CFTR protein, human; D000939:Epitopes; D005456:Fluorescent Dyes; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP