Isoform Selectivity of 3-125I-Iodo-␣-Methyl-LTyrosine Membrane Transport in Human L-Type
Amino Acid Transporters
Naoto Shikano, MS1; Yoshikatsu Kanai, MD2; Keiichi Kawai, PhD3; Jun Inatomi, MD2; Do Kyung Kim, MD2;
Nobuyoshi Ishikawa, MD1; and Hitoshi Endou, MD2
1Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan; 2Department ofPharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan; and 3School of Health Sciences, Faculty ofMedicine, Kanazawa University, Kanazawa, Japan

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 acidtransporters, 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 inthe Na⫹-free uptake solution containing 18.5 kBq of noncarrieradded 125I-IMT. After incubation for 30 min at room temperature,radioactivity of the oocytes was determined. Results: Of the 2hLAT isoforms and h4F2hc-coexpressed X. laevis oocytes, 125IIMT 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 withother 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
J Nucl Med 2003; 44:244 –246

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 clinicallyfor SPECT of tumors (1,2). In this pilot study, we firstcompared 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 ofhuman 4F2 cell-surface antigen (h4F2hc) for system L-likefunctional expression (3,4). Uptake studies of 125I-IMT wereperformed with hLAT1 or hLAT2 and h4F2hc-coexpressed
Xenopus laevis oocytes. Among the amino acid transportsystems described, system L is a Na⫹-independent transportsystem and a major route for providing cells with large neutralamino 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).
MATERIALS AND METHODS
Labeled Compounds
Reagent grade chemicals (Aldrich Chemical Co., Milwaukee,WI) were used in this experiment. 125I-NaI (8.1 ⫻ 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 wasobtained 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 andhLAT2 using T3 RNA polymerase for hLAT1 and hLAT2 inpBluescript 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 ngof h4F2hc cRNA (molar ratio, 1:1) were injected into X. laevisoocytes. The control group consisted of X. laevis oocytes injectedwith water instead of cRNA solution. Uptake of radiolabeledamino acids was measured 2 d after injection in an Na⫹-freeuptake solution (100 mmol/L choline chloride, 2 mmol/L KCl, 1mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L Hepes, 5 mmol/LTris, pH 7.4; incubation for 30 min at room temperature) containing 18.5 kBq/mL 125I-IMT or L-14C(U)-Tyr.
RESULTS

Received Apr. 10, 2002; revision accepted Sep. 25, 2002.
For correspondence or reprints contact: Naoto Shikano, MS, Department ofRadiological Sciences, Ibaraki Prefectural University of Health Sciences,4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan.
E-mail: sikano@ipu.ac.jp

244

Labeling efficiency was ⬎80% and, after purification,
radiochemical purities of 125I-IMT were ⬎95%. Specificradioactivity was ⬎8.1 ⫻ 1019 Bq/mol.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 44 • No. 2 • February 2003

FIGURE 1. Expected structure of functional unit of L-typeamino acid transporter. Human 4F2 heavy chain (CD98;h4F2hc), type II membrane glycoprotein, has molecular weightof approximately 80 kDa. This protein facilitates transport of125I-IMT by forming heterodimer by disulfide bond with lightchain. Light chain has molecular weight of approximately 40kDa. Human LAT1 and hLAT2 (light chains) have putative 12transmembrane domains. Complementary DNAs of these transporters have been cloned.

125I-IMT uptake of X. laevis oocytes via hLAT1-h4F2hc

heterodimer was 5.95-fold higher than uptake of hLAT2h4F2hc heterodimer (P ⬍ 0.005), whereas L-14C(U)-Tyruptake was not significantly different between hLAT1h4F2hc– and hLAT2-h4F2hc– coexpressing X. laevis oocytes (Fig. 2).
DISCUSSION

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 oftransporter expression (3,4). LAT1 exhibits higher affinity(Km ⫽ 20 – 40 ␮mol/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 intranscellular 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 ␮mol/L) but higher capacity and remarkably broad
substrate selectivity, including smaller neutral amino acids
Gly, Ala, Ser, and Thr (4).
Although expression of LAT2 has not been detected intumor cells (4), high expression of LAT1 has been confirmed in numerous types of tumor cells (3). Table 1 also
lists tumor cell types that express LAT1 (3).
In this pilot study, membrane transport of 125I-IMT andthe parent L-14C(U)-Tyr differed in terms of isoform selectivity. Of the 2 human LAT family isoforms, 125I-IMTtransport preferred hLAT1 to hLAT2. The expression ofamino acid transport proteins differs between cell types andis 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 intoamino acid transport. Further studies with transport of125I-IMT and other compounds via LATs are now inprogress. Several studies have used cell lines to investigate 125I-IMT transport and some have reported thatsystems 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, althoughsystem y⫹L cannot yet be excluded, evidence of its
involvement is inconclusive (6). Investigations into iso-FIGURE 2. Comparison of isoform selectivity of 125I-IMT transport (A) and L-14C(U)-Tyr(B) in hLAT1 or hLAT2 and h4F2hc-coexpressing X. laevis oocytes (water-injectedoocytes acted as control). Uptake of radiolabeled amino acids was measured in Na⫹free uptake solution containing 18.5 kBq
125I-IMT or L-14C(U)-Tyr. 125I-IMT transportshowed hLAT1-h4F2hc selectivity. *P ⬍0.05; **P ⬍ 0.005.

LAT ISOFORM SELECTIVITY OF IMT TRANSPORT • Shikano et al.

245

TABLE 1Expression of LAT1 and LAT2 Confirmed in Mammalian Organs and Tumor Cell Types
DistributionOrgans

Tumor cell types

LAT1

LAT2Heart, brain*, placenta*, lung, liver, skeletal muscle, kidney, pancreas*,
spleen, thymus, prostate, testis*, ovary, small intestine, colon,
peripheral leukocytes*, bone marrow*, fetal liver*
Teratocarcinoma*, bladder carcinoma*, lung small cell carcinoma*,uterine cervical carcinoma*, leukemia*, lymphoma*

Brain, placenta*, skeletal muscle,kidney*, testis*, small intestine**High expression.

form selectivity of 125I-IMT transport with other transportsystems will no doubt be undertaken.

Research (grants 9808-3, 0118-1, and 0220-1) and theIbaraki Prefectural University Grants-in-Aid for the Encouragement of Young Scientists 2001 and 2002.

CONCLUSION

Of the 2 heterodimeric complexes of hLAT1– 4F2hc andhLAT2– 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 PrefecturalUniversity) for their technical assistance. This work wassupported by Grants-in-Aid for Scientific Research (grants10770451, 14770498, and 13557075) from the Ministry ofEducation, Science, Sports and Culture of Japan and theJapan Society for Promotion of Science. Financial support
was also provided by Ibaraki Prefectural University Project246

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