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
INSERM U652, 75006 Paris, France; IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France.
IFR58, Institut des Cordeliers, 75006 Paris, France, Universite Paris-Descartes, 75006 Paris, France; CNRS-UPMC UMR7134, 75006 Paris, France.
J Biol Chem. 2007 Nov 16;282(46):33817-33830. doi: 10.1074/jbc.M700195200. 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.
顶端布美他尼敏感的Na(+)-K(+)-2Cl(-)共转运体，称为NKCC2，是肾髓袢升支粗段主要的盐转运途径。NKCC2的表面表达受细胞内蛋白质转运的调控。然而，参与NKCC2细胞内转运的蛋白质伙伴仍不清楚。此外，由于难以在哺乳动物细胞中表达NKCC2蛋白，旨在了解NKCC2翻译后调控的研究受到了阻碍。在这里，我们通过标记NKCC2的N端结构域，能够在肾上皮细胞中表达NKCC2蛋白。为了深入了解NKCC2转运的调控机制，我们以NKCC2的C端尾巴为诱饵，利用酵母双杂交系统筛选NKCC2的相互作用伙伴。醛缩酶B被鉴定为一种主要的新型相互作用蛋白。对肾显微切割小管进行实时PCR显示醛缩酶B在髓袢升支粗段表达。免疫共沉淀和免疫共定位实验证实了肾细胞中NKCC2与醛缩酶的相互作用。生物素化分析表明，醛缩酶的共表达降低了NKCC2的表面表达。在醛缩酶底物1,6-二磷酸果糖存在的情况下，醛缩酶的结合被破坏，醛缩酶的共表达对NKCC2的细胞表面水平没有进一步影响。最后，功能研究表明，醛缩酶诱导的NKCC2在质膜上的下调与其转运活性的降低有关。总之，我们鉴定出醛缩酶B是一种新型的NKCC2结合伙伴，它在调节NKCC2表面表达中起关键作用，从而揭示了一种控制共转运体细胞内转运的新调控机制。此外，本文描述的NKCC2在哺乳动物细胞中的蛋白表达及其通过蛋白质-蛋白质相互作用的调控，可能为研究该共转运体的细胞生物学和转录后调控开辟新的重要途径。
