An Apical PDZ Protein Anchors the Cystic Fibrosis Transmembrane Conductance Regulator to the Cytoskeleton

Abstract

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl؊ channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl ؊ channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl ؊ channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.Cystic fibrosis (CF) 1 is a lethal autosomal recessive disease characterized by defects in epithelial ion transport (1). CF is caused by mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-regulated Cl Ϫ channel at the apical cell surface (1-3). The CF phenotype includes changes in cellular processes distinct from those involving Cl Ϫ transport, including sodium hyperabsorption and abnormalities in the processing of mucins (4 -6). The most common cause of CF are mutations that lead to the formation of an abnormally folded CFTR protein that does not reach the cell surface (2). Even wild type CFTR is inefficiently transported to the cell surface, with up to 70% of the newly synthesized proteins failing to achieve a stable conformation that escapes quality control mechanisms in the endoplasmic reticulum (2,7,8). Knowledge of the protein-protein interactions that are involved in CFTR-mediated regulation of other epithelial transport proteins, and the interactions that control the trafficking, localization, and regulation of CFTR, is incomplete. Recently, the amino terminus of CFTR was shown to interact with syntaxin 1, with implications both for insertion of CFTR into the plasma membrane and regulation of channel activity (9). Other interactions that stabilize CFTR or regulate its function remain to be identified.Compartmentalization of CFTR in a multiprotein complex might regulate CFTR activity by stabilizing the protein at the cell surface or by increasing the efficiency by which kinases and phosphatases control the channel. The presence of such a complex may also explain how CFTR modulates the activity of other epithelial cell transport proteins. A common mechanism to establish multiprotein complexes is via protein-protein interactions w...

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…We localized ezrin and EBP50 to the apical PM of human airway epithelial cells (Short et al, 1998;Mohler et al, 1999;Ingraffea et al, 2002;Kulaksiz et al, 2002). Both proteins colocalized to the microvillus domain of epithelial cells in nasal, tracheal, bronchial, and bronchiolar superficial epithelia and were expressed at equivalent levels in CF and normal epithelia.…”

“…We focused on EBP50 and ezrin localization as these are the most widely studied ERM proteins relevant to positioning CFTR within the PM (Short et al, 1998;Mohler et al, 1999).…”

“…In some experiments, reducing (sodium borohydride) or oxidizing agents (hydrogen peroxide; both from Sigma) were used after the permeabilization step for epitope retrieval. Serial tissue sections were incubated with antibodies against MUC5AC to identify goblet cells, ␤IV-tubulin or tubulin to identify ciliated cells, AQP5 to identify serous cells in gland acini (Kreda et al, 2001), and ezrin and EBP50 to stain the scaffolding complex associated with CFTR (Short et al, 1998;Mohler et al, 1999). In some experiments, CFTR mAbs were coincubated with polyclonal antibodies against AQP5 and EBP50, or a rat mAb against tubulin, followed by fluorescent anti-mouse IgG Fab fragment and AlexaFluor 633 phalloidin plus either fluorescent anti-rabbit IgG, or anti-rat IgG, diluted 1:200, 1:150, 1:200, and 1:200 respectively.…”

“…However, it has recently been suggested that scaffolding proteins of the ezrin-radixin-moesin (ERM) family must be correctly localized at the PM for proper CFTR trafficking and function (Short et al, 1998;Mohler et al, 1999;Huang et al, 2003). Epithelial inflammation and remodeling have been reported to alter ERM protein expression (Laoukili et al, 2001), which could impair localization of ⌬F508 protein to the apical PM.…”

Extensions of PDZ domains as important structural and functional elements

“…Another example of an extension being important for ligand binding has been reported for NHERF1, which is a PDZcontaining protein involved in assembling signaling complexes and regulating the endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) among other functions (Short et al, 1998;Weinman et al, 2006). With a helical extension attached after the canonical domain (as shown in Fig.…”