The Calcineurin Homologous Protein-1 Increases Na+/H+-Exchanger 3 Trafficking via Ezrin Phosphorylation

Abstract

The Na ϩ /H ϩ -exchanger 3 (NHE3) is essential for regulation of Na ϩ transport in the renal and intestinal epithelium. Although changes in cell surface abundance control NHE3 function, the molecular signals that regulate NHE3 surface expression are not well defined. We found that overexpression of the calcineurin homologous protein-1 (CHP1) in opossum kidney cells increased NHE3 transport activity, surface protein abundance, and ezrin phosphorylation. CHP1 knockdown by small interfering RNA had the opposite effects. Overexpression of wild-type ezrin increased both NHE3 transport activity and surface protein abundance, confirming that NHE3 is downstream of ezrin. Expression of a pseudophosphorylated ezrin enhanced these effects, whereas expression of an ezrin variant that could not be phosphorylated prevented the downstream effects on NHE3. Furthermore, CHP1 knockdown reversed the activation of NHE3 by wild-type ezrin but not by the pseudophosphorylated ezrin. Taken together, these results demonstrate that CHP1 increases NHE3 abundance and constitutive function in a manner dependent on ezrin phosphorylation.

Chronic regulation of the renal Na+/H+exchanger NHE3 by dopamine: translational and posttranslational mechanisms

“…Immunocytochemistry was performed where OKP cells were fixed (4% paraformaldehyde in PBS ϫ 20 min) and permeabilized (0.2% saponin, 3% gelatin ϫ 20 min). The cells were then incubated with primary antibody (monoclonal anti-NHE3 3H3) (23,26,32,33,41,68), followed by incubation with FITC-conjugated donkey anti-mouse polyclonal antibodies (Jackson ImmunoResearch Laboratories). Confocal fluorescent images were visualized through a Zeiss ϫ100 objective lens using a Zeiss LSM-410 laser-scanning confocal microscope.…”

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

“…At 90-95% confluence, cells were transiently transfected with human ezrin cDNA mutant constructs (Babich and Di Sole, 2015;Di Sole et al, 2009) by using Lipofectamine 3000 reagent according to the manufacturer's protocol and the experiments were conducted 48 h later. All of these clones behave as dominant mutants over the endogenous protein.…”

NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway

“…The average TER measurement of Transwells in the 16 CFTR K1468X lacking the 12 C-terminal amino acids, 17 inserted into pTM1, was generously provided by Prof. MJ Welsh (University of Iowa, Iowa City, USA). Human ezrin cDNA constructs (wild-type, active (T567D), and negative (T567A)-dominant form) were in the pEGFP-N1 vector as described by Favia et al 15 and Di Sole et al 18 CFTR 1-633 aa was generated as follows: pBQ6.2 plasmid (gently provided by Dr Johanna Rommens, Hospital for Sick Children, Toronto, Canada) containing full-length CFTR was digested with SmaI and BamHI, and the resulting cDNA (first 1430 bp of CFTR) was inserted into pRRL.CMV.MCS.MM vector provided by Dr L Naldini (HS Raffaele Scientific Institute, Milan, Italy). A PCR product corresponding to 1431-1899 bp of CFTR was generated using the following primers: 5 0 -GATAGAAAGAGGACAGTTGTTG-3 0 (forward) and 5 0 -GTGGCTAGCGAGTTCTGAAAATGT-3 0 (reverse) using pBQ6.2 plasmid as template and cloned in pBSMYC2 (pBluescript containing a myc-tag, gently provided by Dr L Naldini) via BamHI and NheI in frame with a myc-tag.…”