Chun Se-Eun, Thakkar Nilay, Oh Yunseok, Park Ji Eun, Han Songhee, Ryoo Gongmi, Hahn Hyunggu, Maeng Sang Hyun, Lim Young-Ran, Han Byung Woo, Lee Wooin
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul, Republic of Korea.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA.
Biochem Pharmacol. 2017 May 1;131:98-105. doi: 10.1016/j.bcp.2017.02.013. Epub 2017 Feb 23.
Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of these important transporter proteins.
有机阴离子转运多肽1B3（OATP1B3）是一种主要的摄取转运体，介导各种内源性底物以及他汀类药物和抗癌药物等临床重要药物的肝脏摄取。然而，控制OATP1B3膜转运的分子机制在很大程度上尚不清楚。最近的几份报告表明，与非恶性肝细胞中表达的OATP1B3相比，存在一种独特的癌症型OATP1B3变体，其缺少N端的28个氨基酸。有趣的是，癌症型OATP1B3变体主要位于细胞质中，这表明OATP1B3的N端区域参与了其膜转运。在本研究中，我们着手通过实验验证OATP1B3 N端区域的重要性，并确定该区域中负责的序列基序。OATP1B3的许多截短或点突变体在HEK293T、HCT-8或MDCK II细胞中瞬时表达，并通过免疫印迹分析它们在细胞质和表面膜组分中的表达。我们的结果表明，OATP1B3的N端序列，特别是在第12至28位氨基酸位置，在其膜转运中可能是不可或缺的。此外，我们使用融合构建体的结果表明，OATP1B3的前50个氨基酸足以实现其膜定位。N端区域在膜定位中的重要性在其他OATP1B亚家族成员OATP1B1和大鼠Oatp1b2中也有体现。我们在N端区域确定负责的氨基酸或结构基序的努力并未确定具有推定二级结构的单个氨基酸或基序。然而，我们目前的研究结果表明，N端区域对于OATP1B亚家族成员的膜定位很重要，并且应该有助于未来对这些重要转运蛋白的调节和膜转运机制的研究。
