Modulation of renal apical organic anion transporter 4 function by two PDZ domain-containing proteins
Modulation of renal apical organic anion transporter 4 function by two PDZ domain-containing proteins
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DOI：
10.1681/asn.2005030306
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发表时间：
2005-12-01
影响因子：
13.6
通讯作者：
Endou, H
中科院分区：
文献类型：
--
作者：
Miyazaki, H;Anzai, N;Endou, H
人有机阴离子转运体4（OAT4）是肾近端小管顶端的一种有机阴离子/二羧酸交换体，介导类固醇硫酸盐（如雌酮 - 3 - 硫酸盐（E1S）和硫酸脱氢表雄酮）的高亲和力转运。在此，通过酵母双杂交试验检测了两种多价PDZ（PSD - 95/大圆盘蛋白/ZO - 1）蛋白PDZK1和NHERF1作为OAT4的相互作用蛋白。这些相互作用需要OAT4的极端C末端区域以及PDZK1的第一和第四PDZ结构域以及NHERF1的第一PDZ结构域。通过表面等离子体共振分析证实了这些相互作用（解离常数分别为：36 nM、1.2 μM和41.7 μM）。体外结合试验和免疫共沉淀研究表明，OAT4野生型而非缺失PDZ基序的突变体直接与PDZK1和NHERF1相互作用。OAT4、PDZK1和NHERF1蛋白显示定位于肾近端小管的顶端膜。与PDZK1或NHERF1的结合增强了OAT4在HEK293细胞中介导的E1S转运活性（1.2 - 1.4倍），并且OAT4的C末端PDZ基序缺失消除了这种效应。转运活性的增强伴随着通过OAT4的E1S转运的最大转运速率（V - max）的改变，并且与OAT4蛋白表面表达水平的增加相关。这项研究表明，OAT4的功能活性通过与PDZK1和NHERF1网络的PDZ相互作用而被调节，并提示OAT4参与肾近端小管中由PDZ支架提供的受调节的顶端有机阴离子处理。
Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K-D: 36 nM, 1.2 mu M, and 41.7 mu M, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E1S transport activities in HEK293 cells (1.2- to 1.4-fold), and the deletion of the OAT4 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by alteration in V-max of E1S transport via OAT4 and was associated with the increased surface expression level of OAT4 protein. This study indicates that the functional activity of OAT4 is modulated through the PDZ interaction with the network of PDZK1 and NHERF1 and suggests that OAT4 is involved in the regulated apical organic anion handling in the renal proximal tubules, provided by the PDZ scaffold.