Utsunomiya-Tate N, Endou H, Kanai Y
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan.
J Biol Chem. 1996 Jun 21;271(25):14883-90. doi: 10.1074/jbc.271.25.14883.
A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.
从小鼠睾丸中分离出一种编码Na⁺依赖性中性氨基酸转运体的cDNA。所编码的蛋白质命名为ASCT2，其氨基酸序列与哺乳动物谷氨酸转运体具有相似性（同一性为40 - 44%），与Na⁺依赖性中性氨基酸转运体ASCT1具有57%的同一性（阿里扎，J.L.，卡瓦诺，M.P.，费尔曼，W.A.，吴，Y.-N.，默多克，G.H.，诺思，R.A.，和阿马拉，S.G.（1993年）《生物化学杂志》268卷，15329 - 15332页；沙夫卡特，S.，塔马拉波，B.K.，基尔伯格，M.S.，普兰纳姆，R.S.，麦克纳马拉，J.O.，瓜达诺 - 费拉兹，A.，和弗雷莫，T.，Jr.（1993年）《生物化学杂志》268卷，15351 - 15355页），还与小鼠脂肪细胞分化相关基因产物AAAT具有94%的同一性（廖，K.，和莱恩，D.（1995年）《生物化学与生物物理研究通讯》208卷，1008 - 1015页）。当在非洲爪蟾卵母细胞中表达时，ASCT2对L - 丙氨酸、L - 丝氨酸、L - 苏氨酸、L - 半胱氨酸和L - 谷氨酰胺等中性氨基酸表现出Na⁺依赖性高亲和力摄取，Km值约为20微摩尔。L - 蛋氨酸、L - 亮氨酸、L - 甘氨酸和L - 缬氨酸也可被ASCT2转运，但亲和力较低。ASCT2的底物选择性是氨基酸转运系统ASC的典型特征，该系统偏好没有庞大或分支侧链的中性氨基酸。ASCT2也以低亲和力（Km = 1.6毫摩尔）转运L - 谷氨酸。降低细胞外pH可增强L - 谷氨酸的转运，这表明L - 谷氨酸是以质子化形式被转运的。与谷氨酸转运体和另一种ASC亚型ASCT1的电生性转运不同，ASCT2介导的氨基酸转运是电中性的。L - 丙氨酸摄取对Na⁺的依赖性符合米氏方程，表明一个Na⁺与一个氨基酸共转运，这与与两个Na⁺偶联的谷氨酸转运体不同。Northern印迹杂交显示ASCT2主要在肾脏、大肠、肺、骨骼肌、睾丸和脂肪组织中表达。ASCT2的功能特性为Na⁺依赖性中性和酸性氨基酸转运体家族的底物结合位点特性及转运机制提供了丰富信息，这将有助于基于ASCT2与该家族其他成员一级结构比较的结构 - 功能分析。
