The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

In the brush border of intestinal and kidney epithelial cells, scaffolding proteins ezrin, Na+-H+ exchanger regulatory factor (NHERF)1 and NHERF2 play important roles in linking transmembrane proteins to the cytoskeleton and assembling signalling regulatory complexes. The last 30 carboxyl residues of NHERF1 and NHERF2 form the EBDs [ezrin, radixin and moesin (ERM)-binding domain]. The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin. The EBRSs are located within 24 and 19 residues immediately upstream of EBDs for NHERF1 and NHERF2 respectively. In OK (opossum kidney) epithelial cells, EBRSs are necessary along with the EBD to distribute NHERF1 and NHERF2 exclusively to the apical domain. Furthermore, phosphorylation of Ser303 located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate. Moreover, increased phosphorylation of Ser303 was functionally significant preventing acute stimulation of NHE3 (Na+-H+ exchanger 3) activity by dexamethasone. * C-ERMAD, : C-terminal ERM-association domain; Caco-2, : colorectal adenocarcinoma cell; EBD, : ERM-binding domain; EBRS, : ERM-binding regulatory sequence; ERM, : ezrin, radixin and moesin; FERM, : band four-point-one, ezrin, radixin, moesin homology domain; IP, : immunoprecipitation; NHE3, : Na+-H+ exchanger 3; NHERF, : Na+-H+ exchanger regulatory factor; NTA, : nitrilotriacetic acid; OK, : opossum kidney; PDZ, : postsynaptic density 95/discs large/zona occludens-1; pHi, : intracellular pH; PIP2, : phosphatidylinositol 4,5-bisphosphate; WGA, : wheat germ agglutinin; WT, : wild-type; ZO-1, : zona occludens-1