Establishment and Characterization of Mammalian Cell Lines Stably Expressing Human L-Type Amino Acid Transporters

Abstract

Abstract. System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na + -independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 -1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14 C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.

Role of amino acid transporter LAT2 in the activation of mTORC1 pathway and the pathogenesis of crescentic glomerulonephritis

“…As there was no available cell line expressing endogenous LAT2 on the plasma membrane, we tested an amino acid starvation experiment by using the S2-hLAT2 cells, a cell line stably expressing recombinant human LAT2. 42 We first confirmed the expression and the plasma membrane localization of LAT2 in this cell line by western blot analysis and surface immunofluorescence staining, respectively. As shown in Figure 8a, western blot analysis of cellular extracts from S2-hLAT2 cell line clearly showed the presence of human LAT2 migrating as a 45-kDa protein.…”

“…42 Cells were cultured in RITC 80-7 medium (IWAKI, Tokyo, Japan) containing 5% FBS, 500 mg/ml G418, 10 mg/ml transferrin, 0.08 U/ml insulin and 10 ng/ml recombinant EGF in a 331C incubator with an atmosphere of 5% CO 2 . Cells cultured on glass cover slips were fixed with 3% formaldehyde in PBS for 15 min at room temperature and reacted with anti-human LAT2 antibodies (1.0 mg/ml) for 60 min at room temperature.…”

The Involvement of L-Type Amino Acid Transporters in Theanine Transport

“…To determine the theanine kinetics of the system L isoforms, S2 cells exogenously expressing hLAT1 and hLAT2 (S2-hLAT1 and S2-hLAT2 respectively) were used. 21) S2-hLAT1 and S2-hLAT2 cells were established previously 21) by transfection of S2 cells with pcDNA3.1 plasmid vectors containing full-length cDNAs of hLAT1 and hLAT2 respectively. They showed high expression of hLAT1 and hLAT2, and had higher 14 C-leucine uptake activity than the S2 cells.…”

“…21) COS1, T24, HuH7, HepG2, and Neuro2A cells were grown in DMEM medium supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 mg/mL) at 37 C in 5% CO 2 . 293A was cultured under same conditions as the COS1 cells, apart from the addition of non-essential amino acids to the culture medium.…”

“…21) On the other hand, moderate inhibition was seen after the addition of glutamine, serine, alanine, valine, and histidine. Basic amino acids such as lysine and arginine, and acidic amino acids such as glutamic acid and aspartic acid, hardly inhibited 3 H-theanine uptake.…”