System L amino acid transporter LAT1 accumulates O-(2-fluoroethyl)-L-tyrosine (FET).

O-(2-fluoroethyl)-L-tyrosine (FET) labeled with fluorine-18 is an important and specific tracer for diagnostics of glioblastoma via positron emission tomography (PET). However, the mechanism of its quite specific accumulation in tumor tissue has not been understood so far. In this work we demonstrate that [(3)H]L-tyrosine is primarily transported by the system L transporter LAT1 in human LN229 glioblastoma cells. FET reduced tyrosine transport, suggesting that it shares the same uptake pathway. More importantly, accumulation of FET was significantly reduced after siRNA-mediated downregulation of LAT1. Xenopus laevis oocytes expressing human LAT1 together with the glycoprotein 4F2hc (necessary to pull LAT-1 to the plasma membrane) exhibited a similar accumulation of FET as observed in glioblastoma cells. In contrast, no accumulation was observed in control oocytes, not overexpressing an exogenous transporter. Because LAT1 works exclusively as an exchanger of amino acids, substrates at one side of the membrane stimulate exchange against substrates at the other side. Extracellular FET stimulated the efflux of intracellular [(3)H]L-leucine, demonstrating that FET is indeed an influx substrate for LAT1. However, FET injected into oocytes was not able to stimulate uptake of extracellular [(3)H]L-leucine, indicating that FET is not a good efflux substrate. Our data, therefore, suggest that FET is trapped within cells due to the asymmetry of its intra- and extracellular recognition by LAT1. If also found for other transporters in tumor cells, asymmetric substrate recognition may be further exploited for tumor-specific accumulation of PET-tracers and/or other tumor-related drugs.

Structure and function of cationic amino acid transporters (CATs).

The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

Voltage dependence of L-arginine transport by hCAT-2A and hCAT-2B expressed in oocytes from Xenopus laevis.

Membrane potential and currents were investigated with the two-electrode voltage-clamp technique in Xenopus laevis oocytes expressing hCAT-2A or hCAT-2B, the splice variants of the human cationic amino acid transporter hCAT-2. Both hCAT-2A- and hCAT-2B-expressing oocytes exhibited a negative extracellular L-arginine concentration ([L-Arg](o))-sensitive membrane potential, additive to the K(+) diffusion potential, when cells were incubated in Leibovitz medium (containing 1.45 mM L-Arg and 0.25 mM L-lysine). The two carrier proteins produced inward and outward currents, which were dependent on the L-Arg gradient and membrane potential. Ion substitution experiments showed that the hCAT-induced currents were independent of external Na(+), K(+), Ca(2+), or Mg(2+). The apparent Michaelis-Menten constant values at -60 mV, obtained from plots of L-Arg-induced currents against [L-Arg](o), were 0.97 and 0.13 mM in oocytes expressing hCAT-2A and hCAT-2B, respectively; maximal currents amounted to -194 +/- 8 and -84 +/- 2 nA, respectively. At saturating [L-Arg](o), the current-voltage relationships of hCAT-2A-expressing oocytes became steeper, yielding an additional conductance up to 2 microS/oocyte, whereas those of hCAT-2B-expressing oocytes were simply shifted to the right, resulting in voltage-independent difference currents. The distinct electrochemical properties of the two isoforms of hCAT-2 are assumed to contribute differentially to the membrane transport and the maintenance of cationic amino acids in various tissues.

Human cationic amino acid transporters hCAT-1, hCAT-2A, and hCAT-2B: three related carriers with distinct transport properties.

In this study, we aimed at analyzing the human homologues of the murine cationic amino acid transporters mCAT-1, mCAT-2A, and mCAT-2B. cDNAs encoding hCAT-1 had been previously reported by two independent groups [Albritton, L.M., et al. (1993) Genomics 12, 430; Yoshimoto, T., et al. (1991) Virology 185, 10]. We isolated cDNAs encoding hCAT-2A and hCAT-2B from a human liver cDNA library and from cDNA derived from the human hepatoma cell line HepG2, respectively. Analyses of the deduced amino acid sequences of both carriers demonstrated 90.9% identity with the respective murine proteins. In their functional domains (42 amino acids), both hCAT-2A and hCAT-2B differ only by one residue from the respective mouse proteins. Thus, CAT-2 proteins demonstrate a higher interspecies conservation than CAT-1 proteins that are overall 86.5% identical between mouse and human and differ by seven residues in the functional domain. The high degree of sequence conservation was reflected by the functional similarity of the human carriers with their mouse homologues. When expressed in Xenopus oocytes, hCAT-1 and hCAT-2B demonstrated transport properties consistent with y+. Unlike the mouse CAT-1 and CAT-2B, whose transport properties could hardly be distinguished, the transport properties of the human CAT-1 and CAT-2B isoforms showed clear differences: hCAT-1 had a 3-fold higher substrate affinity and was more sensitive to trans-stimulation than hCAT-2B. In contrast to the y+ carriers, hCAT-2A exhibited a 10-30-fold lower substrate affinity, a greater maximal velocity, and was much less sensitive to trans-stimulation at physiological substrate concentrations.

Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B.

The inducible human cationic amino acid transporter hCAT-2B was expressed in Xenopus laevis oocytes, and this system was used to test the effect of several NO synthase (NOS) inhibitors and/or L-arginine analogues on L-arginine transport by this y+ carrier. L-NG-Methyl-L-arginine (L-NMA), asymmetrical L-NG, NG-dimethyl-L-arginine (L-ADMA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-NG-nitro-L-arginine (L-NNA), and L-NG-nitro-L-arginine methyl ester (L-NAME) all inhibited the inducible NOS II extracted from RAW 264.7 macrophages induced with bacterial lipopolysaccharide. L-NMA, L-ADMA, and L-NIO also competed with L-arginine for transport by hCAT-2B, whereas L-NNA and L-NAME did not. The two L-arginine analogues, symmetrical NG, NG-dimethyl-L-arginine (L-SDMA) and alpha-amino-delta-isothioureidovaleric acid (AITV), as well as L-lysine, did not block enzymatic activity of NOS II, but did compete for L-arginine transport mediated by hCAT-2B. L-Lysine and L-SDMA were transported efficiently by hCAT-2B and exchanged against intracellular L-arginine, resulting in an L-arginine depletion of the cells. AITV was a much poorer substrate of hCAT-2B and had only little effect on intracellular L-arginine concentrations. These data indicate that substrate recognition differs markedly between the inducible L-arginine transporter hCAT-2B and the inducible NOS II, with different L-arginine analogues having affinity to only one or both of these proteins.