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Brief Communications |
-Methyl-L-Tyrosine Membrane Transport in Human L-Type Amino Acid Transporters
1 Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan
2 Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan
3 School of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa, Japan
ABSTRACT
3-123I-Iodo-
-methyl-L-tyrosine (123I-IMT) has been developed for SPECT of amino acid transport imaging. We examined the isoform selectivity of 125I-IMT transport of the 2 human L-type amino acid transporters, hLAT1 and hLAT2, with human 4F2hc-coexpressed Xenopus laevis oocytes. Methods: An uptake study of 125I-IMT was performed using transporter-expressed X. laevis oocytes. Oocytes were injected with 17.6 ng of hLAT1 or hLAT2 complementary RNA (cRNA) and 7.4 ng of h4F2hc cRNA in a molar ratio of 1:1. Two days after injection, the uptake of 125I-IMT was measured in the Na+-free uptake solution containing 18.5 kBq of noncarrier-added 125I-IMT. After incubation for 30 min at room temperature, radioactivity of the oocytes was determined. Results: Of the 2 hLAT isoforms and h4F2hc-coexpressed X. laevis oocytes, 125I-IMT uptake via hLAT1 was 5.95-fold higher than that via hLAT2 (P < 0.005). Conclusion: 125I-IMT transport was hLAT1 selective. Investigations on the isoform selectivity of 125I-IMT transport with other transporters are anticipated.
Key Words: membrane transport • 3-125I-iodo--methyl-L-tyrosine • human L-type amino acid transporter family • human 4F2hc • Xenopus laevis oocyte
The artificial amino acid 3-123I-iodo--methyl-L-tyrosine (123I-IMT), which is derived from tyrosine, was developed as a functional imaging agent for neutral amino acid transport in the brain and pancreas and has been used clinically for SPECT of tumors (1,2). In this pilot study, we first compared the transporter selectivity of 125I-IMT and L-14C(U)-Tyr in isoforms of human L-type amino acid transporters (LATs) designated hLAT1 and hLAT2 (Fig. 1) (3,4). Human LAT1 and hLAT2 requires a heavy chain of human 4F2 cell-surface antigen (h4F2hc) for system L-like functional expression (3,4). Uptake studies of 125I-IMT were performed with hLAT1 or hLAT2 and h4F2hc-coexpressed Xenopus laevis oocytes. Among the amino acid transport systems described, system L is a Na+-independent transport system and a major route for providing cells with large neutral amino acids, including branched or aromatic amino acids (5). The hypothesis has been proposed that amino acid transporters in transformed cells are upregulated to support high-level protein synthesis for continuous growth and proliferation (5). In cultured human glioma cells, membrane transport of 125I-IMT is dominated by amino acid transport system L (2).
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Labeled Compounds
Reagent grade chemicals (Aldrich Chemical Co., Milwaukee, WI) were used in this experiment. 125I-NaI (8.1 x 1019 Bq/mol) was obtained from Amersham Pharmacia Biotech (Buckinghamshire, U.K.). Noncarrier-added 125I-IMT was prepared by the conventional chloramine-T method as described (1). L-14C(U)-Tyr was obtained from American Radiolabeled Chemicals (St. Louis, MO).
Uptake Studies with X. Laevis Oocytes
As described previously (3,4), in vitro transcription was performed to obtain complementary RNAs (cRNAs) for hLAT1 and hLAT2 using T3 RNA polymerase for hLAT1 and hLAT2 in pBluescript II SK- (Stratagene, La Jolla, CA) linearized with Xho I and T7 RNA polymerase for h4F2hc in plasmid pZL1 (Invitrogen, Carlsbad, CA) linearized with BamHI. For X. laevis oocyte expression studies, 17.6 ng of hLAT1 or hLAT2 cRNA and 7.4 ng of h4F2hc cRNA (molar ratio, 1:1) were injected into X. laevis oocytes. The control group consisted of X. laevis oocytes injected with water instead of cRNA solution. Uptake of radiolabeled amino acids was measured 2 d after injection in an Na+-free uptake solution (100 mmol/L choline chloride, 2 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L Hepes, 5 mmol/L Tris, pH 7.4; incubation for 30 min at room temperature) containing 18.5 kBq/mL 125I-IMT or L-14C(U)-Tyr.
RESULTS
Labeling efficiency was >80% and, after purification, radiochemical purities of 125I-IMT were >95%. Specific radioactivity was >8.1 x 1019 Bq/mol.
125I-IMT uptake of X. laevis oocytes via hLAT1-h4F2hc heterodimer was 5.95-fold higher than uptake of hLAT2-h4F2hc heterodimer (P < 0.005), whereas L-14C(U)-Tyr uptake was not significantly different between hLAT1-h4F2hc- and hLAT2-h4F2hc-coexpressing X. laevis oocytes (Fig. 2).
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LAT1 and LAT2, belonging to the mammalian LAT family, selectively transport neutral amino acids by obligatory exchange mechanisms (3,4). These transporters possess broad substrate selectivity for various neutral amino acids and have different roles. Table 1 shows the distribution of transporter expression (3,4). LAT1 exhibits higher affinity (Km = 20–40 µmol/L) but lower capacity toward Leu, Ile, Phe, Met, Tyr, His, Trp, and Val. D-Isomers of Leu, Phe, and Met are also accepted as substrates (3). The heterodimeric complex of LAT2 and 4F2hc is involved in transcellular transport of neutral amino acids through epithelia and blood-tissue barriers (4). Compared with LAT1, which prefers larger neutral amino acids with branched or aromatic side chains, LAT2 exhibits lower affinity (Km = 30–300 µmol/L) but higher capacity and remarkably broad substrate selectivity, including smaller neutral amino acids Gly, Ala, Ser, and Thr (4).
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In this pilot study, membrane transport of 125I-IMT and the parent L-14C(U)-Tyr differed in terms of isoform selectivity. Of the 2 human LAT family isoforms, 125I-IMT transport preferred hLAT1 to hLAT2. The expression of amino acid transport proteins differs between cell types and is sometimes dependent on the differentiation state of cells (5). Development of isoform-selective artificial amino acids is expected to facilitate research into tumors, cerebral function, and other organs and to provide clues for research into amino acid transport. Further studies with transport of 125I-IMT and other compounds via LATs are now in progress. Several studies have used cell lines to investigate 125I-IMT transport and some have reported that systems L, A, T, and B0,+ (or B0) play a role (2,6). Na+-independent carrier systems such as b0,+ and y+ do not play a role in 125I-IMT uptake (6). However, although system y+L cannot yet be excluded, evidence of its involvement is inconclusive (6). Investigations into isoform selectivity of 125I-IMT transport with other transport systems will no doubt be undertaken.
CONCLUSION
Of the 2 heterodimeric complexes of hLAT1-4F2hc and hLAT2-4F2hc, 125I-IMT transport was hLAT1-4F2hc selective, whereas the parent L-14C(U)-Tyr did not demonstrate isoform selectivity.
ACKNOWLEDGMENTS
The authors thank Tomoaki Murakami, Makoto Hira, Miya Kikuchi, Kohichi Hamazaki, Michiko Nishikido, Mayo Hakamada, and Rinko Sasaki (Ibaraki Prefectural University) for their technical assistance. This work was supported by Grants-in-Aid for Scientific Research (grants 10770451, 14770498, and 13557075) from the Ministry of Education, Science, Sports and Culture of Japan and the Japan Society for Promotion of Science. Financial support was also provided by Ibaraki Prefectural University Project Research (grants 9808-3, 0118-1, and 0220-1) and the Ibaraki Prefectural University Grants-in-Aid for the Encouragement of Young Scientists 2001 and 2002.
FOOTNOTES
Received Apr. 10, 2002; revision accepted Sep. 25, 2002.
For correspondence or reprints contact: Naoto Shikano, MS, Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan.
E-mail: sikano{at}ipu.ac.jp
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-methyl tyrosine transport ([123I]IMT) transport into human Ewing’s sarcoma cells in vitro. Nucl Med Biol. 2001;28:123–128.[Medline]This article has been cited by other articles:
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