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Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association.

ABSTRACT: The heteromeric amino acid transporter glycoprotein subunits rBAT and 4F2hc (heavy chains) form, with different catalytic subunits (light chains), functional heterodimers that are covalently stabilized by a disulphide bridge. Whereas rBAT associates with b(0,+)AT to form the cystine and cationic amino acid transporter defective in cystinuria, 4F2hc associates with other homologous light chains, for instance with LAT1 to form a system L neutral amino acid transporter. To identify within the heavy chains the domain(s) involved in recognition of and functional interaction with partner light chains, chimaeric and truncated forms of rBAT and 4F2hc were co-expressed in Xenopus laevis oocytes with b(0,+)AT or LAT1. Heavy chain-light chain association was analysed by co-immunoprecipitation, and transport function was tested by tracer uptake experiments. The results indicate that the cytoplasmic tail and transmembrane domain of rBAT together play a dominant role in selective functional interaction with b(0,+)AT, whereas the extracellular domain of rBAT appears to facilitate specifically L-cystine uptake. For 4F2hc, functional interaction with LAT1 was mediated by the N-terminal part, comprising cytoplasmic tail, transmembrane segment and neck, even in the absence of the extracellular domain. Alternatively, functional association with LAT1 was also supported by the extracellular part of 4F2hc comprising neck and glycosidase-like domain linked to the complementary part of rBAT. In conclusion, the cytoplasmic tail and the transmembrane segment together play a determinant role for the functional interaction of rBAT with b(0,+)AT, whereas either cytoplasmic or extracellular glycosidase-like domains are dispensable for the functional interaction of 4F2hc with LAT1.

SUBMITTER: Franca R

PROVIDER: S-EPMC1138950 | biostudies-other | 2005 Jun

REPOSITORIES: biostudies-other

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Project description:The cationic amino acid arginine, due to its positive charge, is usually accumulated in the cytosol. Nevertheless, arginine has to be released by a number of cell types, e.g. kidney cells, which supply other organs with this amino acid, or the endothelial cells of the blood-brain barrier which release arginine into the brain. Arginine release in mammalian cells can be mediated by two different transporters, y(+)LAT1 and y(+)LAT2. For insertion into the plasma membrane, these transporters have to be associated with the type-II membrane glycoprotein 4F2hc [Torrents, Estevez, Pineda, Fernandez, Lloberas, Shi, Zorzano and Palacin (1998) J. Biol. Chem. 273, 32437-32445]. The present study elucidates the function and distribution of y(+)LAT2. In contrast to y(+)LAT1, which is expressed mainly in kidney epithelial cells, lung and leucocytes, y(+)LAT2 has a wider tissue distribution, including brain, heart, testis, kidney, small intestine and parotis. When co-expressed with 4F2hc in Xenopus laevis oocytes, y(+)LAT2 mediated uptake of arginine, leucine and glutamine. Arginine uptake was inhibited strongly by lysine, glutamate, leucine, glutamine, methionine and histidine. Mutual inhibition was observed when leucine or glutamine was used as substrate. Inhibition of arginine uptake by neutral amino acids depended on the presence of Na(+), which is a hallmark of y(+)LAT-type transporters. Although arginine transport was inhibited strongly by glutamate, this anionic amino acid was only weakly transported by 4F2hc/y(+)LAT2. Amino acid transport via 4F2hc/y(+)LAT2 followed an antiport mechanism similar to the other members of this new family. Only preloaded arginine could be released in exchange for extracellular amino acids, whereas marginal release of glutamine or leucine was observed under identical conditions. These results indicated that arginine has the highest affinity for the intracellular binding site and that arginine release may be the main physiological function of this transporter.

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Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association.

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