Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator ΔF508
| Autor: | John L. Teem, Ana C. deCarvalho, Lisa J. Gansheroff |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Phenylalanine
Amino Acid Motifs Blotting Western Genetic Vectors Molecular Sequence Data Cystic Fibrosis Transmembrane Conductance Regulator ATP-binding cassette transporter Biology Transfection Models Biological Biochemistry Cystic fibrosis Cell Line Cyclic AMP medicine Animals Humans Amino Acid Sequence ΔF508 Molecular Biology Gene Alleles Dose-Response Relationship Drug Models Genetic Sequence Homology Amino Acid Colforsin Wild type Cell Biology medicine.disease Genistein Molecular biology Cystic fibrosis transmembrane conductance regulator Protein Structure Tertiary Rats Electrophysiology Cyclic nucleotide-binding domain Mutation Mutagenesis Site-Directed Chloride channel biology.protein ATP-Binding Cassette Transporters Electrophoresis Polyacrylamide Gel Chlorine Gene Deletion HeLa Cells Plasmids |
| Zdroj: | Journal of Biological Chemistry. 277:35896-35905 |
| ISSN: | 0021-9258 |
| Popis: | The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif. |
| Databáze: | OpenAIRE |
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