The NHE3 juxtamembrane cytoplasmic domain directly binds ezrin: Dual role in NHE3 trafficking and mobility in the brush border
The NHE3 juxtamembrane cytoplasmic domain directly binds ezrin: Dual role in NHE3 trafficking and mobility in the brush border
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DOI：
10.1091/mbc.e05-09-0843
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发表时间：
2006-06-01
影响因子：
3.3
通讯作者：
Donowitz, Mark
中科院分区：
文献类型：
--
作者：
Cha, Boyoung;Tse, Ming;Donowitz, Mark
根据生理学研究，上皮刷状缘(BB)Na+/H+反转录因子3(NHE3)似乎通过与含有NHERF1和NHERF2的PDZ结构域结合或独立于肌动蛋白细胞骨架而与肌动蛋白细胞骨架联系。我们现在发现NHE3直接与Ezrin结合在其C末端AA 475-589之间的一个位置，该位置独立于PSD95/DLG/zonular occludens-1(PDZ)相互作用结构域。这是一个预测为a螺旋的区域，一侧有正的AA簇(K516、R520和R527)。这些带正电的AA的点突变减少(NHE3双突变体[R520F，R527F])或取消(NHE3三突变体[K516Q，R520F，R527F])Ezrin结合。这些NHE3点突变的功能后果包括以下几点。1)表面量明显减少，NHE3活性下降幅度较大。2)由于新合成的NHE3的胞吐速率和质膜递送率降低，表面表达减少，总表达水平正常，内吞速率略有降低。3)突变体NHE3的质膜半衰期较长，总半衰期正常。4)降低了NHE3双突变体的BB移动率。这些结果表明，NHE3直接或间接地与Ezrin结合，提示前者与1)新合成的NHE3的胞外转运和质膜递送有关，NHE3决定质膜NHE3的数量并部分决定NHE3的活性；2)NHE3的BB迁移率可能增加其从微绒毛到绒毛间隙的转运，可能与NHE3调节的内吞作用有关。
Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be a-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.