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J. Biol. Chem., Vol. 282, Issue 11, 8099-8109, March 16, 2007

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Targeting CAL as a Negative Regulator of F508-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, and Dean R. Madden¹

From the Departments of Biochemistry and ^¶Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, the Department of Physiology, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, and the ^||Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

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 F508-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.

Received for publication, May 11, 2006 , and in revised form, November 30, 2006.

^* This work was supported in part by Cystic Fibrosis Foundation Grants STANTO97R0 (to B. A. S.), MIERKE05G0 (to D. F. M. and D. R. M.), MADDEN06P0 (to D. R. M.), KIVENS04H0 (to A. K.), and the Research Development Program (to W. B. G.), National Institutes of Health Grant 1 P20 RR018787 from the Institutional Development Award (IDeA) Program of the National Center for Research Resources (to D. R. M.), and National Institutes of Health Grants R01 45881 (to B. A. S.) and R01 HL47122 (to W. B. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 7200 Vail Bldg., Hanover, NH 03755. Tel.: 603-650-1164; Fax: 603-650-1128; E-mail: drm0001{at}dartmouth.edu.

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