Wade James B, Liu Jie, Coleman Richard A, Cunningham Rochelle, Steplock Deborah A, Lee-Kwon Whaseon, Pallone Thomas L, Shenolikar Shirish, Weinman Edward J
Dept. of Physiology, 655 W, Baltimore St., Univ. of Maryland, Baltimore, MD 21201, USA.
Am J Physiol Cell Physiol. 2003 Dec;285(6):C1494-503. doi: 10.1152/ajpcell.00092.2003. Epub 2003 Aug 13.
In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.
在表达系统和酵母中，已证实钠/氢交换调节因子（NHERF）-1和NHERF-2可与肾刷状缘膜蛋白NHE3和Npt2相互作用。然而，在小鼠的肾组织中，尽管存在NHERF-2，但NHERF-1对于NHE3的cAMP调节以及Npt2的顶端靶向是必需的，这表明存在另一种特异性顺序。本研究检测了NHERF-1和NHERF-2的亚细胞定位及其与包括NHE3、Npt2和埃兹蛋白在内的靶蛋白的相互作用。野生型小鼠近端小管以独特的模式表达NHERF-1和NHERF-2。NHERF-1在微绒毛中与NHE3、Npt2和埃兹蛋白一起强烈表达。虽然在微绒毛中可微弱检测到NHERF-2，但它主要在富含囊泡的微绒毛基部表达。NHERF-2似乎直接与埃兹蛋白和NHE3相关联，但不与Npt2相关联。NHERF-1参与Npt2的顶端表达，并且在NHERF-1基因敲除小鼠中，其他Npt2结合蛋白的存在并不能完全补偿NHERF-1的缺失。尽管NHERF-1通过埃兹蛋白将NHE3与肌动蛋白细胞骨架连接起来，但NHERF-1的缺失并不会导致细胞结构的普遍破坏。因此，在NHERF-1基因敲除小鼠中看到的Npt2靶向错误可能代表由NHERF-1介导的实现Npt2靶向的途径的特异性破坏。这些发现表明，NHERF异构体的有组织的亚细胞分布可能在介导转运蛋白功能生理控制的特异性相互作用中起作用。
