Targeting CAL as a negative regulator of ΔF508-CFTR cell-surface expression: An RNA interference and structure-based mutagenetic approach

Michael Wolde, Abigail Fellows, Jie Cheng, Aleksandr Kivenson, Bonita Coutermarsh, Laleh Talebian, Katherine Karlson, Andrea Piserchio, Dale F. Mierke, Bruce A. Stanton, William B. Guggino, Dean R. Madden

Research output: Contribution to journalArticle

Abstract

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

Original languageEnglish (US)
Pages (from-to)8099-8109
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number11
DOIs
StatePublished - Mar 16 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Wolde, M., Fellows, A., Cheng, J., Kivenson, A., Coutermarsh, B., Talebian, L., Karlson, K., Piserchio, A., Mierke, D. F., Stanton, B. A., Guggino, W. B., & Madden, D. R. (2007). Targeting CAL as a negative regulator of ΔF508-CFTR cell-surface expression: An RNA interference and structure-based mutagenetic approach. Journal of Biological Chemistry, 282(11), 8099-8109. https://doi.org/10.1074/jbc.M611049200