Elucidation of the cell-and organ-specific transport mechanisms of organic anion transporters

阐明有机阴离子转运蛋白的细胞和器官特异性转运机制

基本信息

批准号：
17590120
负责人：
KATSURA Toshiya
金额：
$ 2.3万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In the epithelial tissues such as small intestine, kidney and liver, various organic anion transporters with distinct characteristics were localized to the brush-border and basolateral membranes, and play an important role in determining the unidirectional transport (absorption or secretion) of drugs. However, the mechanism of plasma membrane targeting of organic anion transporters was not fully understood. In the present study, organic anion transporter OAT-K was selected as a model transporter, and we constructed the expression vector in which enhanced green fluorescent protein (EGFP) was fused to the N-terminus of OAT-K. EGFP-OAT-K fusion protein was stably expressed in MDCK cells and its biosynthesis and cell surface targeting were examined. Our data showed that OAT-K was modified by post-translational processing and its C-terminal portion was targeted to the apical membrane. It was suggested that processing of OAT-K occurred at the endoplasmic reticulum and that its N-terminal portion was degraded by proteasomal pathway.Other organic anion transporters, OAT1 and OAT3, are abundantly expressed in the human kidney, and therefore we examined the mechanisms concerning their regulation of expression by promoter analysis. It was demonstrated that a cAMP-response element exists in the promoter region of OAT3 gene. Transcription factors, CREB-1 and ATF-1, regulate the constitutive and inducible expression via a cAMP-response element, while OAT1 gene was shown to be regulated by hepatocyte nuclear factor-4α. We also succeeded in cloning of MATE1 and MATE2-K, the H^+/organic cation antiporters expressed in the brush-border membrane, and demonstrated their tissue distribution and functional characteristics.We will compare the mechanisms of membrane targeting between brush-border type and basolateral type of organic ion transporters in the future.

在小肠，肾脏和肝脏等上皮组织中，具有不同特征的各种有机阴离子转运蛋白被定位于刷子和基底外侧机制，并且在确定药物的单向运输（吸收或秘密）中起着重要作用。但是，尚未完全了解有机阴离子转运蛋白的质膜靶向的机理。在本研究中，选择有机阴离子转运蛋白燕麦-K作为模型转运蛋白，我们构建了表达载体，其中增强的绿色荧光蛋白（EGFP）融合到OAT-K的N末端。 EGFP-AAT-K融合蛋白在MDCK细胞中稳定表达，并检查其生物合成和细胞表面靶向。我们的数据表明，燕麦K是通过翻译后处理来改变的，其C末端部分针对顶端膜。有人提出，燕麦k的加工发生在内质网处，其N末端部分通过蛋白酶体途径降解。事实证明，OAT3基因的启动子区域中存在cAMP响应元件。转录因子CREB-1和ATF-1通过CAMP响应元件调节构型和诱导的表达，而OAT1基因被证明受肝细胞核因子-4α的调节。我们还成功地将MATE1和MATE2-K克隆，在刷子膜中表达的H^+/有机阳离子抗焦剂，并证明了它们的组织分布和功能特性。我们将比较未来有机离子移植者的刷子析出型和基底外侧类型的膜靶向。