title: Association of domains within the cystic fibrosis transmembrane conductance regulator.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel composed of two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and an R domain. To understand how these domains interact, we expressed various constructs of CFTR containing membrane-spanning and/or cytosolic domains either separately or together. We then tested for functional association of these domains using the SPQ halide-efflux assay or physical association using coimmunoprecipitation experiments. Coexpression of the amino-terminal half (MSD1, NBD1, and the R domain) and the carboxy-terminal half (MSD2 and NBD2) of CFTR generated functional Cl- channel activity whereas expression of either alone did not give a signal with the SPQ assay. This result suggests that the two halves associate in the membrane. Using domain-specific antibodies, we found that either half of CFTR could coimmunoprecipitate the other, suggesting a physical association. Coimmunoprecipitation persisted between halves missing the NBDs, the R domain, or the amino-terminal tail. Moreover, constructs from MSD1 containing only the first and second transmembrane sequences and intervening extracellular loop were sufficient for interaction with MSD2. These data suggest that interactions between the two membrane-spanning domains of CFTR may mediate association between the two halves of the protein.
authors.affiliation: Department of Internal Medicine, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
authors.forename: L S
authors.initials: LS
authors.lastname: Ostedgaard
authors.forename: D P
authors.initials: DP
authors.lastname: Rich
authors.forename: L G
authors.initials: LG
authors.lastname: DeBerg
authors.forename: M J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator