E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells

Although it is generally recognized that cystic fibrosis transmembrane cond uctance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding mo tif at its COOH terminus, the identity of the PDZ domain protein(s) that in teract with CFTR is uncertain, and the functional impact of this interactio n 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 doma in protein. The PDZ binding motif located at the COOH terminus of CFTR inte racts preferentially with the second PDZ domain of E3KARP, with nanomolar a ffinity. 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 mono layers shows that E3KARP and CFTR are co-localized at the apical membrane d omain. We also found that ezrin associates with E3KARP in vivo. Go-expressi on of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimul ated CFTR Cl- currents. These results support the concept that E3KARP funct ions as a scaffold protein that links CFTR to ezrin, Since ezrin has been s hown previously to function as a protein kinase A anchoring protein, we sug gest 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 actin-binding protein, the formation of a CFT R E3KARP ezrin complex may be important also in stabilizing CFTR at the api cal membrane domain of airway cells.