期刊
BIOLOGY OF THE CELL
卷 104, 期 11, 页码 658-676出版社
WILEY
DOI: 10.1111/boc.201100074
关键词
Kidney; Membrane Transport; Protein sorting; trafficking; targeting
类别
资金
- Italian grant PRIN [20078ZZMZW]
- Fondo per gli Investimenti della Ricerca di Base-Rete Nazionale di Proteomica [RBRN07BMCT_009]
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 protein (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.
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