title: Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.
abstract: Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.
authors.affiliation: Department of Cell Biology, University of Alabama at Birmingham, AL 35294, USA.
authors.forename: Asta
authors.initials: A
authors.lastname: Jurkuvenaite
authors.forename: Karoly
authors.initials: K
authors.lastname: Varga
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Nowotarski
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Kirk
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.forename: John P
authors.initials: JP
authors.lastname: Clancy
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Bebok
authors.forename: James F
authors.initials: JF
authors.lastname: Collawn
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D019921:Biotinylation; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000494:pharmacology; D047468:Immunoprecipitation; D008954:Models, Biological; D009154:Mutation; D018408:Patch-Clamp Techniques; D010641:Phenotype; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011806:Quinolinium Compounds / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014443:Tyrosine; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D001120:Arginine