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Cytoskeletal keratin 18 regulates the cell surface expression of CFTR channels via direct binding
Author(s) -
Duan Yuanyuan,
Yang Pingfang,
Zhang Wei,
Sun Ying,
Wang Dong,
Cuppoletti John,
Wang Haixia,
Wu Zhenguo,
Huang Pingbo
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a548-c
Subject(s) - cystic fibrosis transmembrane conductance regulator , microbiology and biotechnology , chemistry , pdz domain , colocalization , hek 293 cells , cystic fibrosis , biology , biochemistry , genetics , gene
Recent studies have shown that several proteins could bind to the cystic fibrosis transmembrane conductance regulator (CFTR) C‐terminus and modulate its surface expression and channel gating, but interactions found to date do not account for all the known functions of the C‐terminus, suggesting that as yet unidentified proteins that bind to this region of CFTR and regulate the properties of the channel. To search for such proteins, we performed yeast two hybrid assays and identified as a binding partner of the CFTR C‐terminus keratin 18 (K18), an intermediate filament protein which has been long been though to simply provide mechanical resilience in simple epithelial cells. The binding sites of CFTR and K18 were identified as the region lying 1407aa‐1436aa of CFTR and the coil‐2 domain of K18 by yeast two hybrid assays. The interaction of K18 with CFTR was further confirmed with GST pull‐down assays and colocalization studies by confocal microscopy. Cell surface biotinylation and patch‐clamp studies in HEK293T cells overexpressing K18 and CFTR indicated that K18 expression robustly increases surface CFTR expression. These results were further verified with endogenous CFTR and K18 in airway epithelial Calu‐3 cells by combining RNA interference and Ussing chamber techniques. Our data suggest a novel regulatory mechanism for CFTR channel function. Supported by RGC grant HKUST6468/05M.