Identification of moesin as NKCC2-interacting protein and analysis of its functional role in the NKCC2 apical trafficking.
BACKGROUND
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.
Department of Biosciences, Biotechnologies and Pharmacological Sciences, University of Bari, 70126 Bari, Italy. monica.carmosino@uniba.it
Monica
M
Carmosino
Federica
F
Rizzo
Giuseppe
G
Procino
Lello
L
Zolla
Anna Maria
AM
Timperio
Davide
D
Basco
Claudia
C
Barbieri
Silvia
S
Torretta
Maria
M
Svelto
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D055785:Gene Knockdown Techniques; D007668:Kidney / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000276:immunology / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013552:Swine / Q000378:metabolism
D008840:Microfilament Proteins; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; C071058:moesin