Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells
Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells
复制标题
DOI：
10.1074/jbc.274.3.1519
复制
发表时间：
1999-01-15
影响因子：
4.8
通讯作者：
You, GF
中科院分区：
文献类型：
--
作者：
Kuze, K;Graves, P;You, GF
有机阴离子转运蛋白在从肾脏清除各种有机阴离子（包括内源性化合物、外源性物质及其代谢物）中发挥重要作用，从而防止其在体内的潜在毒性作用。本研究的目的是扩展我们以前的研究的功能特性和翻译后修饰的小鼠肾脏有机阴离子转运蛋白（mOAT），在哺乳动物细胞系统，COS-7细胞。转运蛋白介导的对氨基马尿酸（PAH）摄取是饱和的，对丙磺舒敏感，可被多种有机阴离子抑制，包括维生素、抗高血压药物、抗肿瘤药物和抗炎药物，衣霉素（Tunicamycin，一种天冬酰胺连接糖基化的抑制剂）可显著抑制PAH的转运活性。免疫荧光提供的证据表明，大多数蛋白质仍然在衣霉素处理的细胞内室。焦碳酸二乙酯（DEPC），组氨酸残基特异性试剂，完全阻断PAH的运输。用过量的未标记PAH预处理细胞，DEPC的抑制作用得到显著保护（90%），这表明组氨酸残基可能接近PAH结合位点。最后，在出生后的小鼠肾脏原位mRNA定位进行了研究。在整个发育过程中，在近端小管中观察到表达。我们的结论是，COS-7细胞可能是有用的药理学和分子生物学研究这种载体。碳水化合物部分是mOAT正确转运至质膜所必需的，组氨酸残基似乎对转运功能很重要。
Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and posttranslational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, antihypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs, Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.