The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus
The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus
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DOI：
10.1074/jbc.m406724200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Endou, H
中科院分区：
文献类型：
--
作者：
Anzai, N;Miyazaki, H;Endou, H
尿酸阴离子交换剂 URAT1 是有机阴离子转运蛋白 (OAT) 家族的成员，可调节人体血尿酸水平，是促尿酸排泄剂和抗尿酸排泄剂的靶标（Enomoto, A.、Kimura, H.、Chairoungdua, A.、Shigeta, Y.、Jutabha, P.、Cha, S. H.、Hosoyamada, M.、Takeda, M.、Sekine, T.、 Igarashi, T.、Matsuo, H.、Kikuchi, Y.、Oda, T.、Ichida, K.、Hosoya, T.、Shimotaka, K.、Niwa, T.、Kanai, Y. 和 Endou, H. (2002) Nature 417, 447-452)。 URAT1 仅在肾脏中表达，被认为参与肾小管尿酸盐重吸收。我们发现，在酵母双杂交筛选中，含有多价 PDZ（PSD-95、果蝇圆盘大蛋白、小带闭合蛋白 1）结构域的蛋白 PDZK1 与 URAT1 相互作用。这种相互作用需要 URAT1 的 PDZ 基序位于其最胞内 C 端区域，以及通过酵母双杂交测定、体外结合测定和表面等离子共振分析（K-D = 1.97-514 nM）鉴定的 PDZK1 的第一、第二和第四 PDZ 结构域。免疫共沉淀研究表明，野生型 URAT1（而非其缺乏 PDZ 基序的突变体）直接与 PDZK1 相互作用。在肾近端肾小管细胞的顶膜处观察到 URAT1 和 PDZK1 的共定位。 URAT1 与 PDZK1 的结合增强了 HEK293 细胞中的尿酸盐转运活性（1.4 倍），而 URAT1 C 端 PDZ 基序的删除消除了这种作用。转运活性的增强伴随着通过 URAT1 的尿酸盐转运 V-max 的显着增加，并且与稳定表达 URAT1（转染 PDZK1）的 HEK293 细胞中 URAT1 蛋白表面表达水平的增加有关。综上所述，本研究表明 PDZK1 在调节肾近曲小管顶膜中 URAT1 介导的尿酸盐转运的功能活性中具有新作用。
The urate-anion exchanger URAT1 is a member of the organic anion transporter ( OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002) Nature 417, 447-452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K-D = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wildtype URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V-max of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.