NHE Exchanger Disruption After Short- and Long-Term TLR Activation

By CO-IP, full-length NHERF2 bound more ezrin than NHERF1.NHERF2 also outcompeted NHERF1 in a competitive ezrin binding assay.In contrast, NHERF1-EBD outperformed NHERF2-EBD by the same competitive assay.PD assays showed that the short non-conserved sequences upstream of EBDs increased the interaction between ezrin and EBDs to different extents in NHERF1 and 2. These non-conserved sequences are designated as the ERM binding regulatory domain (EBRD).EBDs alone were localized to both microvilli and cytosol in OK cell.However, the combination of EBD and EBRD was exclusively localized to microvilli, fully mimicking full-length NHERF1/2 proteins.The EBRD of NHERF2 contains two phosphorylation residues, Ser303 and Thr305.Phosphomimetic mutations S303D, T305D and T305E reduced NHERF2-ezrin interaction.S303D mutation also dislocated NHERF2 from microvilli to cytosol.Conclusions: 1) Though NHERF1-EBD's affinity for ezrin is higher than that of NHERF2-EBD, full-length NHERF1 has a lower ezrin binding affinity than NHERF2.2) The short sequences upstream of EBD form a functional EBRD domain, which enhances ezrin/NHERF1 and 2 binding.3) EBRD is necessary along with the EBD to distribute NHERF1/2 exclusively to the microvilli.4) Phosphorylation of Ser-303 and perhaps Thr-305 diminishes NHERF2-EBRD function, reduces ezrin binding to NHERF2, and partially dislocates NHERF2 into the cytosol.This is the first function identified for NHERF2 phosphorylation.