Abstract
We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.
Highlights
We have isolated a cDNA from rat small intestine that encodes a novel Na؉-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property
We have demonstrated that co-expression of LAT-1 and 4F2 heavy chain (4F2hc) results in the functional expression of system L activity. 4F2 antigen is a heterodimeric protein composed of two subunits, an ϳ80-kDa glycosylated heavy chain and an ϳ40-kDa nonglycosylated light chain [19, 20]. 4F2hc is an integral membrane protein with a single membrane-spanning domain classified as a type II membrane protein [21]
Structural Features of L-type amino acid transporter-2 (LAT-2)—A cDNA clone with a 4117base pair insert was isolated from a rat small intestine cDNA library
Summary
CDNA Cloning—The cDNA for a human expressed sequence tag (EST) Xenopus oocyte expression studies and uptake measurements were performed as described [5, 32]. The Xenopus oocyte expression of LAT-1 and 4F2hc was performed as described [18]. Efflux Measurements—For the efflux measurements, 14C-labeled Lleucine and other amino acids were preloaded by incubating the oocytes expressing LAT-2 and 4F2hc or LAT-1 and 4F2hc in the Naϩ-free uptake solution containing 150 (for LAT-2) or 20 M (for LAT-1) L-[14C]leucine for 30 min. Radiolabeled amino acids used were the following: U-14C-labeled compounds for L-leucine, L-alanine, L-serine, L-threonine, L-glutamine, L-isoleucine, L-valine, L-phenylalanine, L-tyrosine, L-histidine, L-aspartate, L-glutamate, L-lysine, L-arginine, L-proline, and L-cystine, [1-14C] glycine, L-[methyl-14C]methionine, and L-[side chain-3-14C]tryptophan from NEN Life Science Products, Inc.; and L-[1-14C]cysteine, L-[G-3H] asparagine, D-[1-14C]leucine, D-[1-14C]phenylalanine, and D-[1-14C] serine from American Radiolabeled Chemicals, Inc. For the measurements of the uptake and the efflux of radiolabeled amino acids in the present study, six to eight oocytes were used for each data point. C6 glioma cells were provided by the Health Science Research Resources Bank, Japan Health Sciences Foundation
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