NKCC2 Surface Expression in Mammalian Cells
NKCC2 Surface Expression in Mammalian Cells
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哺乳动物细胞中的 NKCC2 表面表达
DOI：
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发表时间：
2007
影响因子：
4.8
通讯作者：
K. Laghmani
中科院分区：
文献类型：
--
作者：
B. Benziane;Sylvie Demaretz;N. Defontaine;N. Zaarour;L. Cheval;Soline Bourgeois;C. Klein;M. Froissart;A. Blanchard;M. Paillard;G. Gamba;P. Houillier;K. Laghmani
顶端布美他尼敏感的Na+- k +- 2cl -共转运蛋白，称为NKCC2，是肾厚升肢中主要的盐转运途径。NKCC2表面表达受细胞内蛋白转运的调控。然而，参与NKCC2细胞内转运的蛋白伙伴仍然未知。此外，由于NKCC2蛋白难以在哺乳动物细胞中表达，旨在了解NKCC2翻译后调控的研究受到了阻碍。通过标记NKCC2蛋白的n端结构域，我们能够在肾上皮细胞中表达NKCC2蛋白。为了深入了解NKCC2的转运调控，我们以NKCC2的c端尾部为诱饵，筛选了NKCC2与酵母双杂交系统的互作伙伴。醛缩酶B是一种显性的新型相互作用蛋白。肾微解剖小管实时荧光定量PCR检测显示增厚升肢中醛缩酶B的表达。共免疫沉淀和共免疫定位实验证实了nkcc2 -醛缩酶在肾细胞中的相互作用。生物素化实验显示醛缩酶的共表达降低了NKCC2表面的表达。在醛缩酶底物、果糖1,6-二磷酸存在时，醛缩酶的结合被破坏，醛缩酶共表达对NKCC2的细胞表面水平没有进一步的影响。最后，功能研究表明醛缩酶诱导的质膜上NKCC2的下调与其运输活性的降低有关。总之，我们发现醛缩酶B是一种新的NKCC2结合伙伴，在NKCC2表面表达的调节中起关键作用，从而揭示了一种新的调控共转运体细胞内运输的调控机制。此外，本文所述的NKCC2蛋白在哺乳动物细胞中的表达及其通过蛋白-蛋白相互作用的调控，可能为研究共转运体的细胞生物学和转录后调控开辟新的重要途径。
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.
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通过针对变性酶的抗血清鉴定醛缩酶 B 的细胞内降解中间体。
DOI：
10.1073/pnas.82.18.6114
发表时间：
1985
影响因子：
11.1
作者：
Reznick,AZ;Rosenfelder,L;Shpund,S;Gershon,D
通讯作者：
Gershon,D
缺磷会抑制 OK 细胞中 NH4 的转运。
DOI：
10.1016/0005-2736(93)90214-k
发表时间：
1993
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Chen,JG;Kempson,SA
通讯作者：
Kempson,SA
DOI：
10.1073/pnas.91.6.2201
发表时间：
1994-03-15
影响因子：
11.1
作者：
XU, JC;LYTLE, C;FORBUSH, B
通讯作者：
FORBUSH, B
DOI：
10.1073/pnas.91.10.4544
发表时间：
1994-05-10
影响因子：
11.1
作者：
PAYNE, JA;FORBUSH, B
通讯作者：
FORBUSH, B
影响因子：
19.6
作者：
Kaplan, MR;Plotkin, MD;Hebert, SC
通讯作者：
Hebert, SC