Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.
Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties.
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DOI：
10.1254/jphs.12124fp
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发表时间：
2012
影响因子：
3.5
通讯作者：
Kanai Y
中科院分区：
文献类型：
--
作者：
Khunweeraphong N;Nagamori S;Wiriyasermkul P;Nishinaka Y;Wongthai P;Ohgaki R;Tanaka H;Tominaga H;Sakurai H;Kanai Y
L系统是细胞摄取中性氨基酸的主要运输系统。在系统L转运蛋白中，L型氨基酸转运蛋白1 （LAT1）负责癌细胞的营养摄取，而L型氨基酸转运蛋白2 （LAT2）负责非癌细胞的转运蛋白。在这项研究中，我们建立了HEK293细胞系，稳定表达高水平的人LAT1和LAT2形成异源二聚体，细胞具有天然的人4F2hc。我们发现l-[14C]丙氨酸是检测LAT2功能的合适底物，而l-[14C]亮氨酸则用于LAT1。通过l-[14C]丙氨酸修饰LAT2，我们首次直接评价了人类LAT2在哺乳动物细胞中表达的功能，并获得了其可靠的动力学。利用α-烷基氨基酸，包括α-甲基丙氨酸和α-乙基丙氨酸，我们证明了α-烷基干扰与LAT2的相互作用。这些具有较高实用优势的细胞系将有助于筛选和分析化合物，以开发可用于癌症诊断和治疗的lat1特异性药物。我们建立细胞系的策略也适用于其他具有重要治疗意义的异二聚体转运体。
System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that l-[14C]alanine is an appropriate substrate to examine the function of LAT2, whereas l-[14C]leucine is used for LAT1. By using l-[14C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-l-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.
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影响因子：
8.8
作者：
Kaira, K.;Oriuchi, N.;Imai, H.;Shimizu, K.;Yanagitani, N.;Sunaga, N.;Hisada, T.;Tanaka, S.;Ishizuka, T.;Kanai, Y.;Endou, H.;Nakajima, T.;Mori, M.
通讯作者：
Mori, M.
影响因子：
6.4
作者：
Kaira, Kyoichi;Oriuchi, Noboru;Mori, Masatomo
通讯作者：
Mori, Masatomo
影响因子：
50.3
作者：
Ishimoto, Takatsugu;Nagano, Osamu;Saya, Hideyuki
通讯作者：
Saya, Hideyuki
影响因子：
3.3
作者：
Kido, Y;Tamai, I;Tsuji, A
通讯作者：
Tsuji, A
影响因子：
3.8
作者：
GRAHAM, FL;SMILEY, J;NAIRN, R
通讯作者：
NAIRN, R