E3KARP Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells

Abstract

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na ؉ /H ؉ exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/ NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl ؊ currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actinbinding protein, the formation of a CFTR⅐E3KARP⅐ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

Ezrin-radixin-moesin–binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia

“…Importantly, these studies indicate that disruption of PDZ domain interactions abolishes the cAMP-dependent Cl Ϫ secretory response in a CFTR-expressing epithelial cell. In other expression studies, EBP50 has been implicated in directly modifying CFTR Cl Ϫ channel activity 45 and directing the cellular distribution of CFTR. 46 Consequently, it will be important to further characterize the effects of PDZ1 expression on the location and function of CFTR as well as other cAMP-regulated anion channels.…”

“…13,45 In both hepatocytes and cholangiocytes, it is of specific interest to note that the apical expression of EBP50 is paralleled by the apical expression of an ERM protein (Figs. 2, 3, 4, 6, and 8).…”

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…NHERF Potentiates the PKA-dependent Activation of CFTR-NHERF may facilitate CFTR activation at the cell membrane by inducing conformational changes, promoting CFTR phosphorylation by anchoring the catalytic subunit of PKA/protein kinase C to the vicinity of the channel (28,29,31), and/or increasing the channel number (32,34). To examine whether NHERF can have a modulatory effect on the channel activity in intact cells, we compared the effect of NHERF and D1 domain on the PKA-stimulated whole cell chloride current activity.…”

“…Channel activation is facilitated by a conformational change of CFTR induced by binding to the multivalent NHERF and 70-kDa CFTR-associated protein, as found in excised inside-out patches (13,30). Furthermore, NHERF may promote the phosphorylation of CFTR by tethering PKA and protein kinase C⑀ to the vicinity of the channel (28,31).…”

“…The D1 and D2 domains bind with nanomolar affinity to the PDZbinding motifs of CFTR ( 1477 DTRL) and to the C terminus of other transport proteins, signaling molecules, and receptors (18,(22)(23)(24)(25)(26). The ERM-binding domain tethers the complex via ezrin to cytoskeletal elements in a phosphorylation-dependent manner and to the catalytic and regulatory subunit of PKA (19,(27)(28)(29).…”