Establishment and characterization of mammalian cell lines stably expressing human L-type amino acid transporters.
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 alpha-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.
Morimoto
Emiko
E
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Japan.
Kanai
Yoshikatsu
Y
Kim
Do Kyung
DK
Chairoungdua
Arthit
A
Choi
Hye Won
HW
Wempe
Michael F
MF
Anzai
Naohiko
N
Endou
Hitoshi
H
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009865:Oocytes; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; C419196:LAT2 protein, human; D027283:Large Neutral Amino Acid-Transporter 1; D012333:RNA, Messenger; D007930:Leucine