We report here on the primary structure and functional characteristics of the protein responsible for the system A amino acid transport activity that is known to be expressed in most human tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from the human hepatoma cell line HepG2. Human ATA2 (hATA2) consists of 506 amino acids and exhibits a high degree of homology to rat ATA2. hATA2-specific mRNA is ubiquitously expressed in human tissues. When expressed in mammalian cells, hATA2 mediates Na+-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li+-intolerant. The Na+:amino acid stoichiometry is 1:1.
We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50-56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na(+)-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li(+) tolerant, and pH sensitive. The transport mechanism involves the influx of Na(+) and amino acids coupled to the efflux of H(+), resulting in intracellular alkalization. Proline, alpha-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.
ATB(0,+) [SLC6A14 (solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of approximately 250 muM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that alpha-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with alpha-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.
We evaluated the potential of the Na ϩ -and Cl Ϫ -coupled amino acid transporter ATB 0,ϩ as a delivery system for amino acidbased prodrugs. Immunofluorescence analysis indicated that ATB 0,ϩ is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of -carboxyl derivatives of aspartate and ␥-carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB 0,ϩ , several of the aspartate and glutamate derivatives inhibited glycine transport via ATB 0,ϩ . Direct evidence for ATB 0,ϩ -mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB 0,ϩ heterologously, to aspartate -benzyl ester as a model derivative induced inward currents in a Na ϩ -and Cl Ϫ -dependent manner with a Na ϩ /Cl Ϫ /aspartate -benzyl ester stoichiometry of 2:1:1. ATB 0,ϩ transported not only the -carboxyl derivatives of aspartate and the ␥-carboxyl derivatives of glutamate but also valacyclovir, which is an ␣-carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB 0,ϩ was demonstrable in both heterologous expression systems. This process was dependent on Na ϩ and Cl Ϫ . The ability of ATB 0,ϩ to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB 0,ϩ has significant potential as a delivery system for amino acid-based drugs and prodrugs. ATB 0,ϩ is a Na ϩ -and Cl Ϫ -coupled amino acid transporter that is energized by transmembrane gradients of Na ϩ and Cl Ϫ as well as by the membrane potential (Palacin et al., 1998;Ganapathy et al., 2001. It belongs to the neurotransmitter transporter gene family (SLC6) (Chen et al., 2003). Among the currently known mammalian amino acid transporters, ATB 0,ϩ is the only transporter with a very broad substrate specificity that is driven by a Na ϩ gradient and a Cl Ϫ gradient. This transporter recognizes neutral as well as cationic amino acids as substrates. In addition to the surprisingly broad substrate specificity of ATB 0,ϩ with regard to the naturally occurring L-amino acids, this transporter can also transport various D-amino acids (Hatanaka et al., 2002), nitric-oxide synthase inhibitors , and carnitine and its esters . Unlike most other amino acid transporters that exhibit a broad expression pattern, Northern blot analysis has revealed that ATB 0,ϩ transcripts are detectable primarily in the colon, lung, and mammary gland (Sloan and Mager, 1999).The fact that ATB 0,ϩ recognizes neutral and cationic amino acids, but not anionic amino acids as substrates, indicates that the presence of a negative charge in the side chain of amino acids prevents their recognition by the transporter as substrates. The presence of an unchar...
We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including alpha-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na(+)-coupled manner and the transport of cationic amino acids in a Na(+)-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y(+)L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.
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