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Identification of moesin as NKCC2‐interacting protein and analysis of its functional role in the NKCC2 apical trafficking
Author(s) -
Carmosino Monica,
Rizzo Federica,
Procino Giuseppe,
Zolla Lello,
Timperio Anna Maria,
Basco Davide,
Barbieri Claudia,
Torretta Silvia,
Svelto Maria
Publication year - 2012
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/boc.201100074
Subject(s) - moesin , radixin , immunoprecipitation , exocytosis , ezrin , microbiology and biotechnology , biology , transport protein , endocytosis , transporter , secretion , cytoskeleton , biochemistry , cell , gene
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.