Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane.
CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700-10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.
Ramjeesingh
Mohabir
M
Programme in Structural Biology and Biochemistry, Physiology Department, Faculty of Medicine, Research Institute, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada M5G 1X8.
Kidd
Jackie F
JF
Huan
Ling Jun
LJ
Wang
Yanchun
Y
Bear
Christine E
CE
D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000737:chemistry; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator