NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B


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NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B
J Biol Chem
Benziane B., Demaretz S., Defontaine N., Zaarour N., Cheval L., Bourgeois S., Klein C., Froissart M., Blanchard A., Paillard M., Gamba G., Houillier P., Laghmani K.
0021-9258 (Print) 0021-9258 (Linking)
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Benziane, Boubacar
Demaretz, Sylvie
Defontaine, Nadia
Zaarour, Nancy
Cheval, Lydie
Bourgeois, Soline
Klein, Christophe
Froissart, Marc
Blanchard, Anne
Paillard, Michel
Gamba, Gerardo
Houillier, Pascal
Laghmani, Kamel
Research Support, Non-U.S. Gov't
2007/09/13 09:00
J Biol Chem. 2007 Nov 16;282(46):33817-30. Epub 2007 Sep 11.
Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.
Animals, Biotinylation, Cell Membrane/metabolism, Epithelial Cells/*metabolism, Fructose-Bisphosphate Aldolase/*chemistry, *Gene Expression Regulation, Kidney/*metabolism, Male, Mice, Mice, Inbred C57BL, Models, Biological, Protein Binding, Protein Interaction Mapping, Protein Structure, Tertiary, Protein Transport, Sodium-Potassium-Chloride Symporters/*physiology, Solute Carrier Family 12, Member 1, Two-Hybrid System Techniques
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03/03/2016 17:49
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