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Biol Cell. 2012 Nov;104(11):658-76. doi: 10.1111/boc.201100074. Epub 2012 Jul 26.

Identification of moesin as NKCC2-interacting protein and analysis of its functional role in the NKCC2 apical trafficking.

Author information

  • 1Department of Biosciences, Biotechnologies and Pharmacological Sciences, University of Bari, 70126 Bari, Italy. monica.carmosino@uniba.it

Abstract

BACKGROUND INFORMATION:

The renal Na(+) -K(+) -2Cl(-) co-transporter (NKCC2) is expressed in kidney thick ascending limb cells, where it mediates NaCl re-absorption regulating body salt levels and blood pressure.

RESULTS:

In this study, we used a well-characterised NKCC2 construct (c-NKCC2) to identify NKCC2-interacting proteins by an antibody shift assay coupled with blue native/SDS-PAGE and mass spectrometry. Among the interacting proteins, we identified moesin, a protein belonging to ezrin, eadixin and moesin family. Co-immunoprecipitation experiments confirmed that c-NKCC2 interacts with the N-terminal domain of moesin in LLC-PK1 cells. Moreover, c-NKCC2 accumulates in intracellular and sub-apical vesicles in cells transfected with a moesin dominant negative green fluorescent protien (GFP)-tagged construct. In addition, moesin knock-down by short interfering RNA decreases by about 50% c-NKCC2 surface expression. Specifically, endocytosis and exocytosis assays showed that moesin knock-down does not affect c-NKCC2 internalisation but strongly reduces exocytosis of the co-transporter.

CONCLUSIONS:

Our data clearly demonstrate that moesin plays a critical role in apical membrane insertion of NKCC2, suggesting a possible involvement of moesin in regulation of Na(+) and Cl(-) absorption in the kidney.

Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

PMID:
22708623
[PubMed - indexed for MEDLINE]
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