Isoform Selectivity of 3-¹²⁵I-Iodo-α-Methyl-l-Tyrosine Membrane Transport in Human L-Type Amino Acid Transporters

MATERIALS AND METHODS

RESULTS

Labeling efficiency was >80% and, after purification, radiochemical purities of ¹²⁵I-IMT were >95%. Specific radioactivity was >8.1 × 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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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-injected oocytes 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 transport showed hLAT1-h4F2hc selectivity. *P < 0.05; **P < 0.005.

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 of transporter expression (3,4). LAT1 exhibits higher affinity (K_m = 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 (K_m = 30–300 μmol/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 in tumor 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 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 B^0,+ (or B⁰) play a role (2,6). Na⁺-independent carrier systems such as b^0,+ 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.