Identification of a Membrane Protein, LAT-2, That Co-expresses with 4F2 Heavy Chain, an L-type Amino Acid Transport Activity with Broad Specificity for Small and Large Zwitterionic Amino Acids

Abstract

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta > > brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or ϳ286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.Last year, three amino acid transporter cDNAs (LAT-1, y ϩ LAT-1, and y ϩ LAT-2) 1 were identified as subunits of the heavy chain of the cell surface antigen 4F2 (4F2hc, also named CD98) (1-3). These subunits co-express amino acid transport activity with 4F2hc in oocytes (i.e. system L for LAT-1, and system y ϩ L for y ϩ LAT-1 and y ϩ LAT-2) (1-4). The role of this family of proteins in amino acid transport has recently been demonstrated by the fact that mutations in the y ϩ LAT-1 gene cause lysinuric protein intolerance, an inherited amino aciduria due to a defective renal reabsorption mechanism of dibasic amino acids (5, 6). The structural and functional similarities between 4F2hc and its homologous protein rBAT suggest that a member of this family of subunits might be the subunit of rBAT needed to fully express the amino acid transport system b o,ϩ activity (reviewed in Refs. 7 and 8). After the identification of rBAT as the Type I cystinuria gene (9), this subunit is a good candidate for non-Type I cystinuria (7). A search throughout gene data bases suggests that there may be as many as four new human members of the family of subunits of 4F2hc and rBAT.Kanai and co-workers (1) identified rat LAT-1 (also known as TA1) by co-expression cloning with 4F2hc in oocytes. The coexpressed transport activity shows clear characteristic...

Basolateral LAT-2 Has a Major Role in the Transepithelial Flux of L-Cystine in the Renal Proximal Tubule Cell Line OK

“…Moreover, among the heteromeric amino acid transporters that mediate exchange of neutral amino acids, only LAT-2 transport activity, but not LAT-1, asc-1 and asc-2, is expressed in the basolateral domain of OK cells. This is in full agreement with the expression of these transporters in the epithelial cells of the renal proximal tubule; LAT-1 is barely expressed in human and mouse kidney (38,39), asc-1 is not expressed in kidney (28), asc-2 is localized in collecting ducts in mouse kidney (29), and only LAT-2 is conspicuously expressed in the epithelial cells of the renal proximal tubule and the small intestine (21)(22)(23)40).…”

“…This K m value is in the range reported for this amino acid via the transport activity elicited by human LAT-2 and 4F2hc cRNAs in Xenopus oocytes (22).…”

“…Therefore, the direction of exchange via LAT-2/4F2hc for a given amino acid is not easily predictable and would depend on its concentration and affinity relative to other substrates of the transporter on either side of the plasma membrane. In kidney, LAT-2 is expressed in the epithelial cells of the upper part of the proximal tubule, close to the glomerulus (21,22). In this part of the proximal tubule the luminal amino acid concentration corresponds to that of the glomerulus filtrate, and it is close to that of the plasma.…”

“…The following criteria were used to detect LAT-2 activity: (1) L-alanine is transported via LAT-2, asc-1, and asc-2 (22,23,28,29), but it is not transported via LAT-1 (30,31); (2) transport of L-alanine via LAT-2, but not via asc-1 and asc-2, is inhibited by BCH and L-tyrosine (23) To check the amino acid exchanger activity of the LAT-2-related L-system (21,22,24), polarized cells were loaded on the basolateral and apical membranes with the radioactive substrate (L-[ 3 H] leucine or L-[ 3 H] alanine), and the efflux across the basolateral membrane was measured. The efflux of Lleucine or L-alanine trans-stimulated by LAT-2 substrates was mostly sodium independent.…”

“…LAT-2 is expressed in the basolateral plasma membrane of the renal epithelial cells of the upper part of the proximal tubule (S1 and S2 segments) (21), where most of amino acid reabsorption occurs (1). LAT-2/4F2hc co-expressed in Xenopus oocytes show exchange of neutral amino acids of any size (21)(22)(23)(24). These characteristics suggest that LAT-2 may play a role in renal reabsorption, but which amino acids use this transporter for net efflux or influx in the context of a renal epithelial cell is unknown.…”