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Am J Physiol Cell Physiol (January 2, 2002). doi:10.1152/ajpcell.00337.2001
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Articles in PresS, published online ahead of print January 2, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00337.2001
Submitted on July 20, 2001
Accepted on November 27, 2001

Domain-Domain Associations in Cystic Fibrosis Transmembrane Conductance Regulator

Wenlan Wang1, Zhaoping He1, Thomas J O'Shaughnessy1, John J Rux2, and William W Reenstra3*

1 Clinical Research, Alfred I. duPont Hospital for Children, Wilmington, DE, USA
2 Wistar Institute, Philadelphia, PA, USA
3 Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA, USA

* To whom correspondence should be addressed. E-mail: reenstra{at}mail.med.upenn.edu.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires PKA-dependent phosphorylation of an intracellular regulatory domain (R-domain), and ATP hydrolysis at the nucleotide binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified and refolded his- and GST-tagged cytoplasmic domains of CFTR. ATP binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching and binding among all pair wise combinations of R-domain, NBD1 and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50 likely sites of interaction between NBD1 and NBD2 were identified. Our data supports a model where NBDs form dimers with the ATP binding sites at the domain-domain interface.




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