Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression

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 ⌬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 CAL⅐CFTR 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.

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

“…In the Golgi, modulation of the function or abundance of PDZ-domain containing proteins has been shown to correct ⌬F508CFTR trafficking (10,22,23). Similarly, EBP50 has been implicated in r⌬F508CFTR biosynthetic processing.…”

MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

“…Our data demonstrated that the binding of MAST205 and CFTR inhibited the PDZ-based binding between CAL and CFTR. It has been reported that overexpression of CAL leads to a dramatic decrease at the plasma membrane pool of both wild type CFTR and F508del-CFTR through promoting lysosomal degradation of CFTR (14,17). Endogenous CAL limits F508del-CFTR half-life, and knockdown of CAL expression level increases the surface pool of functional F508del-CFTR in human airway epithelial cells (17).…”

“…It has been reported that overexpression of CAL leads to a dramatic decrease at the plasma membrane pool of both wild type CFTR and F508del-CFTR through promoting lysosomal degradation of CFTR (14,17). Endogenous CAL limits F508del-CFTR half-life, and knockdown of CAL expression level increases the surface pool of functional F508del-CFTR in human airway epithelial cells (17). Recent studies suggested that TC10, a small Rho-GTPase interacting with CAL and inhibiting CFTR-CAL binding, increased CFTR expression at the plasma membrane (36).…”

“…Overexpression of CAL has been shown to reduce the half-life of CFTR at the plasma membrane and promote CFTR degradation in lysosomes. In contrast, silencing of CAL has been shown to restore function to CFTR and rescue the mutant F508del-CFTR (14,16,17). The CAL-induced reduction in CFTR expression and distribution in cells can be restored by the overexpression of NHERF-1, acting through a competition mechanism for binding to the PDZ motif of CFTR (18).…”

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) Increases the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Airway Epithelial Cells by Phosphorylating Shank2E Protein

“…We and others have shown that plasma membrane abundance of CFTR is regulated by a number of PDZ proteins, including NHERF1/EBP50, NHERF2, and CFTR-associated ligand (1,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). A common feature of these proteins is the presence of a PDZ (PSD-95, Dlg, ZO-1) domain, a protein-protein interaction module that engages the C terminus of CFTR.…”