PDZK1 directly regulates the function of organic cation/carnitine transporter OCTN2
PDZK1 directly regulates the function of organic cation/carnitine transporter OCTN2
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DOI：
10.1124/mol.104.002212
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Tsuji, A
中科院分区：
文献类型：
--
作者：
Kato, Y;Sai, Y;Tsuji, A
阳离子外源性物质的尿排泄被认为是由肾小管基底外侧膜和刷状缘膜上表达的有机阳离子转运蛋白（OCT和OCTN）家族介导的，尽管这些转运蛋白靶向每个膜的分子机制知之甚少。在这里，检查这些转运蛋白的细胞表面表达和功能的调节机制，我们评估了这些转运蛋白与几种PDZ蛋白的相互作用。使用OCT和OCTN的重组C-末端蛋白的下拉研究确定了顶端转运蛋白OCTN 1和OCTN 2与PDZK 1、肠道和肾脏富集的PDZ蛋白和Na+/H+交换调节因子2（也称为E3 KARP、SIP-1或TKA-1）的特异性相互作用，但基底外侧转运蛋白OCT 1和OCT 2不存在。酵母双杂交和下拉研究都表明OCTN 1和OCTN 2中最后四个氨基酸的相互作用是必需的。PDZK 1与OCTN 2的C末端的相互作用也在使用肾刷状缘膜囊泡的下拉研究中得到证实。免疫组织化学分析显示，PDZK 1和OCTN 2共定位于肾刷状缘膜。最后，用PDZK 1双重转染OCTN 2刺激OCTN 2对其内源性底物肉毒碱的摄取，并且这种增加可以通过转运能力增加6倍来解释。对于缺失最后四个氨基酸的OCTN 2没有观察到这种增加。表面蛋白的生物素化研究揭示了PDZK 1对OCTN 2的细胞表面表达的最小影响。目前的研究结果是第一个通过与C末端直接相互作用将PDZK 1鉴定为OCTN 2的功能调节剂。
Urinary excretion of cationic xenobiotics is believed to be mediated by organic cation transporter (OCT and OCTN) families expressed on both basolateral and brush-border membranes of renal tubules, although the molecular mechanisms for targeting of these transporters to each membrane are poorly understood. Here, to examine the regulatory mechanisms for cell-surface expression and function of these transporters, we evaluated the interaction of these transporters with several PDZ proteins. A pull-down study using recombinant C-terminal proteins of OCTs and OCTNs identified a specific interaction of apical transporters OCTN1 and OCTN2, but not basolateral transporters OCT1 and OCT2, with PDZK1, intestinal and kidney-enriched PDZ protein, and Na+/H+ exchanger regulatory factor 2 (also called E3KARP, SIP-1, or TKA-1). Both yeast two-hybrid and pull-down studies suggested a requirement of the last four amino acids in OCTN1 and OCTN2 for the interaction. The interaction of PDZK1 with the C terminus of OCTN2 was also confirmed in a pull-down study using kidney brush-border membrane vesicles. Immunohistochemical analysis revealed that both PDZK1 and OCTN2 are colocalized in brush-border membranes of the kidney. Finally, double transfection of OCTN2 with PDZK1 stimulated the uptake by OCTN2 of its endogenous substrate carnitine, and this increase could be accounted for by the 6-fold increase in transport capacity. Such an increase was not observed for OCTN2 with deletion of the last four amino acids. Biotinylation study of surface proteins revealed minimal effect of PDZK1 on cell-surface expression of OCTN2. The present findings are the first to identify PDZK1 as a functional regulator of OCTN2 through direct interaction with the C terminus.