Abstract
Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.
Highlights
Ezrin is a member of the highly evolutionarily conserved ERM (Ezrin, Radixin and Moesin) protein family
We found that ezrin binding to PIP2 and its phosphorylation at threonine 567 are both involved in controlling Na+/H+ exchanger-3 (NHE3) activity under baseline conditions (Figure A in S1 File)
Ezrin-PIP2- cellular localization changed when the threonine 567 was mutated to aspartic acid, Ezrin-PIP2- T/D (Fig 1D); Ezrin-PIP2T/D localization resembled that of Ezrin-T/D indicating that mimicking ezrin phosphorylation was sufficient to secure a predominant cell surface expression of ezrin
Summary
Ezrin is a member of the highly evolutionarily conserved ERM (Ezrin, Radixin and Moesin) protein family It provides a regulated linkage between the plasma membrane and the actin cytoskeleton [1, 2]. Upon reaching the plasma membrane, ezrin engages a number of membrane-associated proteins through its N-terminal domain [13, 14] One such protein is the Na+/H+ exchanger-3 (NHE3) that is expressed on the brush border membrane of renal and intestinal epithelial cells [15, 16] and is of major importance in mediating Na+, Cl-, HCO3- and fluid absorption in these epithelia [17,18,19]. We found that ezrin binding to PIP2 and its phosphorylation at threonine 567 are both involved in controlling NHE3 activity under baseline conditions (Figure A in S1 File). Further studies are necessary to understand how CHP1 controls ezrin phosphorylation (Figure B in S1 File, pathway 2)
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