Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface
| Autor: | Steven Molinski, Andrew J. Miles, Donghe Yang, Christine E. Bear, Stephanie Chin, Paul D. W. Eckford, Bonnie A. Wallace |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
spectroscopy Cystic Fibrosis Mutation Missense Cystic Fibrosis Transmembrane Conductance Regulator macromolecular substances bcs Biochemistry 03 medical and health sciences Protein Domains Humans Protein kinase A Molecular Biology synchrotron radiation circular dichroism Ion channel biology phosphorylation Chemistry Molecular Bases of Disease Cell Biology Cyclic AMP-Dependent Protein Kinases cysteine-mediated cross-linking Cystic fibrosis transmembrane conductance regulator enzymes and coenzymes (carbohydrates) Cytosol HEK293 Cells 030104 developmental biology Amino Acid Substitution Membrane protein Cyclic nucleotide-binding domain ion channel biology.protein Biophysics Phosphorylation cystic fibrosis transmembrane conductance regulator (CFTR) Intracellular |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m116.762633 |
| Popis: | Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. |
| Databáze: | OpenAIRE |
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