Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2
Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2
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DOI：
10.1113/jphysiol.2002.026500
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发表时间：
2003-01-15
影响因子：
5.5
通讯作者：
Ganapathy, V
中科院分区：
文献类型：
--
作者：
Chen, Z;Fei, YJ;Ganapathy, V
从人肠细胞系Caco-2中克隆了H+偶联氨基酸转运蛋白（hPAT 1）的人直系同源物，并在哺乳动物细胞异源表达系统中评估了其功能特征。克隆的hPAT 1由476个氨基酸组成，与大鼠PAT 1具有85%的同源性。在通过北方印迹检测的各种人体组织中，PAT 1 mRNA最主要地在肠道中表达。当在哺乳动物细胞中异源表达时，hPAT 1介导α-（甲氨基）异丁酸（MeAIB）的转运。cDNA诱导的运输是Na+-独立的，但由向内定向的H+梯度供能。hPAT 1与甘氨酸、L-丙氨酸C、L-脯氨酸、α-氨基异丁酸（AIB）和γ-氨基丁酸（GABA）相互作用，如直接运输测量和以MeAIB作为运输底物的竞争实验所证明的。hPAT 1还识别丙氨酸和脯氨酸的D-异构体。对于丝氨酸和半胱氨酸，尽管L-异构体没有与hPAT 1相互作用到任何显着的程度，相应的D-异构体被认为是底物。对于脯氨酸和丙氨酸，L-和D-异构体的亲和力相似。然而，与半胱氨酸和丝氨酸，D-异构体显示6- 8倍高的亲和力hPAT 1比相应的L-异构体。hPAT 1的这些功能特征与先前描述的Caco-2细胞中H+偶联氨基酸转运系统的功能特征非常相似。此外，在用天然Caco-2细胞和用异源表达系统中的hPAT 1测量的各种氨基酸抑制H+-偶联MeAIB转运的相对效力之间存在高度相关性（r（2）= 0.93）。免疫定位研究表明，PAT 1只在Caco-2细胞的顶膜表达。这些数据表明，hPAT 1是负责H+-偶联的氨基酸转运表达在Caco-2细胞的顶膜。
The human orthologue of the H+-coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco-2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of alpha-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na+-independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, L-alaninC, L-proline, alpha-aminoisobutyrate (AIB) and gamma-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the D-isomers of alanine and proline. With serine and cysteine, though the L-isomers did not interact with hPAT1 to any significant extent, the corresponding D-isomers were recognized as substrates. With proline and alanine, the affinity was similar for L- and D-isomers. However, with cysteine and serine, the D-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding L-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+-coupled amino acid transport system in Caco-2 cells. Furthermore, there was a high degree of correlation (r(2) = 0.93) between the relative potencies of various amino acids to inhibit the H+-coupled MeAIB transport measured with native Caco-2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 cells. These data suggest that hPAT1 is responsible for the H+-coupled amino acid transport expressed in the apical membrane of Caco-2 cells.