Matsuo Hirotaka, Kanai Yoshikatsu, Kim Ju Young, Chairoungdua Arthit, Kim Do Kyung, Inatomi Jun, Shigeta Yasuhiro, Ishimine Hisako, Chaekuntode Sophapun, Tachampa Kittipong, Choi Hye Won, Babu Ellappan, Fukuda Jun, Endou Hitoshi
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
J Biol Chem. 2002 Jun 7;277(23):21017-26. doi: 10.1074/jbc.M200019200. Epub 2002 Mar 20.
We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.
我们鉴定出一种新的不依赖钠离子的酸性氨基酸转运体，命名为AGT1（天冬氨酸/谷氨酸转运体1）。AGT1与不依赖钠离子的小中性氨基酸转运体Asc（asc型氨基酸转运体）-2具有最高的序列相似性（48%的一致性），Asc-2是异二聚体氨基酸转运体家族的成员之一，推测与未知的重链相关（Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390 - 49399）。负责异二聚体氨基酸转运体家族转运体（轻链）与重链亚基之间二硫键形成的半胱氨酸残基在AGT1中是保守的。由于单独表达或与已知的重链4F2hc（4F2重链）或rBAT（与b(0,+)-氨基酸转运体相关）共表达的AGT1均未诱导出功能活性，我们构建了AGT1与4F2hc或rBAT连接的融合蛋白。这些融合蛋白被分选到质膜上，并表现出对酸性氨基酸的不依赖钠离子的转运活性。与结构上与AGT1相关的不依赖钠离子的胱氨酸/谷氨酸转运体xCT不同，AGT1不接受胱氨酸、高半胱氨酸和L-α-氨基己二酸，对天冬氨酸和谷氨酸表现出高亲和力，这表明AGT1侧链结合位点中的负电荷识别位点与xCT相比更靠近α-碳结合位点。AGT1的信使核糖核酸主要在肾脏中表达。在小鼠肾脏中，AGT1蛋白存在于近端直小管和远端曲小管的基底外侧膜中。在蛋白质印迹分析中，在非还原条件下AGT1被检测为一条高分子量条带，而在还原条件下该条带迁移至对应于AGT1单体的40 kDa条带，这表明AGT1通过二硫键与其他蛋白质结合。AGT1和Asc-2的发现建立了异二聚体氨基酸转运体家族的一个新亚组，其成员不与4F2hc或rBAT结合，而是与其他未知的重链结合。
