title: NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B.
abstract: 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.
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Boubacar
authors.initials: B
authors.lastname: Benziane
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Demaretz
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Defontaine
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Nancy
authors.initials: N
authors.lastname: Zaarour
authors.affiliation: IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; CNRS-UPMC UMR7134, 75006 Paris, France.
authors.forename: Lydie
authors.initials: L
authors.lastname: Cheval
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Soline
authors.initials: S
authors.lastname: Bourgeois
authors.affiliation: IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Klein
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; AP-HP, Departement de Physiologie, Hopital Europeen Georges Pompidou, 75015 Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Froissart
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; AP-HP, Departement de Physiologie, Hopital Europeen Georges Pompidou, 75015 Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Blanchard
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; AP-HP, Departement de Physiologie, Hopital Europeen Georges Pompidou, 75015 Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Paillard
authors.affiliation: Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran and Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Tlalpan, Mexico City 14000, Mexico.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Gamba
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; AP-HP, Departement de Physiologie, Hopital Europeen Georges Pompidou, 75015 Paris, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Houillier
authors.affiliation: INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France. Electronic address: Kamel.Laghmani@bhdc.jussieu.fr.
authors.forename: Kamel
authors.initials: K
authors.lastname: Laghmani
mesh_terms: D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D005786:Gene Expression Regulation; D007668:Kidney / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*; D064466:Solute Carrier Family 12, Member 1; D020798:Two-Hybrid System Techniques
chemical_list: C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D005634:Fructose-Bisphosphate Aldolase