Sodium-dependent Neutral Amino Acid Research Articles

Arsenic trioxide induces unfolded protein response in vascular endothelial cells

Chronic arsenic exposure has been linked to endothelial dysfunction and apoptosis. We investigate the involvement of unfolded protein response (UPR) signaling in the arsenic-mediated cytotoxicity of the SVEC4-10 mouse endothelial cells. The SVEC4-10 cells underwent apoptosis in response to As2O3 dose- and time-dependently, accompanied by increased accumulation of calcium, and activation of caspase-3. These phenomena were completely inhibited by α-lipoic acid (LA), which did not scavenge ROS over-production, but were only partially or not ameliorated by tiron, a potent superoxide scavenger. Moreover, arsenic activated UPR, leading to phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2α), induction of ATF4, and processing of ATF6. Treatment with arsenic also triggered the expression of endoplasmic reticulum (ER) stress markers, GRP78 (glucose-regulated protein), and CHOP (C/EBP homologous protein). The activation of eIF2α, ATF4 and ATF6 and expression of GRP78 and CHOP are repressed by both LA and tiron, indicating arsenic-induced UPR is mediated through ROS-dependent and ROS-independent pathways. Arsenic also induced ER stress-inducible genes, BAX, PUMA (p53 upregulated modulator of apoptosis), TRB3 (tribbles-related protein 3), and SNAT2 (sodium-dependent neutral amino acid transporter 2). Consistent with intracellular calcium and cell viability data, ROS may not be important in arsenic-induced death, because tiron did not affect the expression of these pro-apoptotic genes. In addition, pretreatment with salubrinal, a selective inhibitor of eIF2α dephosphorylation, enhanced arsenic-induced GRP78 and CHOP expression and partially prevented arsenic cytotoxicity in SVEC4-10 cells. Taken together, these results suggest that arsenic-induced endothelial cytotoxicity is associated with ER stress, which is mediated by ROS-dependent and ROS-independent signaling.

Characterization of feline ASCT1 and ASCT2 as RD-114 virus receptor

RD-114 virus is a replication-competent feline endogenous retrovirus (ERV). RD-114 virus had been thought to be xenotropic; however, recent findings indicate that RD-114 virus is polytropic and can infect and grow efficiently in feline cells. Receptor(s) for RD-114 virus has not been identified and characterized in cats. In this study, we confirmed that two feline sodium-dependent neutral amino acid transporters (ASCTs), fASCT1 and fASCT2, function as RD-114 virus receptors. By chimeric analyses of feline and murine ASCTs, we revealed that extracellular loop 2 of both fASCT1 and fASCT2 determines the susceptibility to RD-114 virus. Further, we revealed ubiquitous expression of these genes, consistent with the general metabolic role of the ASCT molecules. Our study indicates that RD-114 virus may reinfect tissues and cells in cats, once the virus is activated. Implications of the involvement of RD-114 virus in feline oncogenesis are also discussed.

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Conserved Residues Serine 225 and Methionine 229 Attenuate the Sodium Transport Responses in the Sodium-Coupled Neutral Amino Acid Transporter, SNAT2

Sodium dependent neutral amino acid transporter (SNAT) 2, a member of the solute carrier (SLC) 38 family, system A, mediates the coupled transport of sodium and small neutral amino acids across a cell's membrane. There has been proposed structural homology between SNAT2 and the sodium/betaine symporter, BetP. We mutated two conserved amino acids that have been shown to be ligands involved in BetP sodium binding: serine-225 and methionine-229. Both residues are found in the predicted TMD 5. The functional consequences of the S225A mutation are a slight reduction in the apparent affinity for alanine, and a dramatic reduction of the apparent sodium affinity. M229A also shows a reduced apparent affinity for alanine. These results demonstrate there is involvement of these residues in the sodium coordination and/or amino acid interaction with SNAT2. It also shows the involvement of TMD 5, which previously had not been thought to be involved with sodium binding in SNAT2.

Functional RNA Interference (RNAi) Screen Identifies System A Neutral Amino Acid Transporter 2 (SNAT2) as a Mediator of Arsenic-induced Endoplasmic Reticulum Stress

Exposure to the toxic metalloid arsenic is associated with diabetes and cancer and causes proteotoxicity and endoplasmic reticulum (ER) stress at the cellular level. Adaptive responses to ER stress are implicated in cancer and diabetes; thus, understanding mechanisms of arsenic-induced ER stress may offer insights into pathogenesis. Here, we identify genes required for arsenite-induced ER stress response in a genome-wide RNAi screen. Using an shRNA library targeting ∼20,000 human genes, together with an ER stress cell model, we performed flow cytometry-based cell sorting to isolate cells with defective response to arsenite. Our screen discovered several genes modulating arsenite-induced ER stress, including sodium-dependent neutral amino acid transporter, SNAT2. SNAT2 expression and activity are up-regulated by arsenite, in a manner dependent on activating transcription factor 4 (ATF4), an important mediator of the integrated stress response. Inhibition of SNAT2 expression or activity or deprivation of its primary substrate, glutamine, specifically suppressed ER stress induced by arsenite but not tunicamycin. Induction of SNAT2 is coincident with the activation of the nutrient-sensing mammalian target of rapamycin (mTOR) pathway, which is at least partially required for arsenite-induced ER stress. Importantly, inhibition of the SNAT2 or the System L transporter, LAT1, suppressed mTOR activation by arsenite, supporting a role for these transporters in modulating amino acid signaling. These findings reveal SNAT2 as an important and specific mediator of arsenic-induced ER stress, and suggest a role for aberrant mTOR activation in arsenic-related human diseases. Furthermore, this study demonstrates the utility of RNAi screens in elucidating cellular mechanisms of environmental toxins.

Canine ASCT1 and ASCT2 are functional receptors for RD-114 virus in dogs

All domestic cats carry an infectious endogenous retrovirus termed RD-114 virus. Recently, we and others found that several live-attenuated vaccines for dogs were contaminated with infectious RD-114 virus. In this study, we confirmed that the RD-114 virus efficiently infected and proliferated well in canine primary kidney cells, as well as three tested canine cell lines. Further, we identified canine ASCT1 and ASCT2, sodium-dependent neutral amino acid transporters, as RD-114 virus receptors. Canine ASCT2 also acts as a functional receptor for simian retrovirus 2, a pathogenic retrovirus that induces immunodeficiency in rhesus macaques. Identification of the canine receptor for RD-114 virus will help in evaluating the risk from vaccines contaminated by the virus.

A Low-Protein Diet during Gestation in Rats Activates the Placental Mammalian Amino Acid Response Pathway and Programs the Growth Capacity of Offspring1–3

Placental efficiency is a predictor of fetal growth and development, which is also controlled by maternal gestational health and diet. The present study investigated the effects of a gestational low-protein diet on offspring growth capacity as well as the diet’s contribution to altered expression of placental genes associated with the mammalian amino acid response (AAR) pathway. To assess these outcomes, timed-pregnant Sprague Dawley rats were fed a control (C) diet with 18% protein or a low-protein (LP) diet with 9% protein throughout gestation (Expt. 1) or throughout gestation and lactation (Expt. 2). Placentas were collected during natural delivery and quantitative RT-PCR and Western-blot analyses were performed to determine placental mRNA and protein levels. By the end of the lactation period, offspring of dams fed the LP diet had stunted growth in both experiments. mRNA expression of target genes in the AAR pathway, such as activating transcription factor-3 (Atf3), asparagine synthetase (Asns), and Sodium-dependent neutral amino acid transporter-2 (Snat2), was greater in placentas of rats fed the LP diet compared with controls, as were placental ATF4 and p-eIF2α protein levels. The increase in mRNA expression of AAR pathway-associated genes was correlated with the stunting of offspring growth (Atf3: R2 = 0.32, P = 0.086; Asns: R2 = 0.44, P < 0.05; Snat2:R2 = 0.33, P = 0.084). Our study showed that the mammalian AAR pathway in placenta is upregulated by a maternal low-protein diet and this activation may act as a cue for the fetus to develop an adaptive response suited to their predicted postnatal environment, i.e. a more favorable phenotype for their survival.

Placental Amino Acid Transport and Placental Leptin Resistance in Pregnancies Complicated by Maternal Obesity

Hypothesis and study objectives We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity. Materials and methods Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 ( p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p ≤ 0.05. The primary outcome measure was placental SNAT activity. Results The obese group had decreased placental SNAT activity ( p = 0.005), maternal hyperleptinemia ( p = 0.01) and decreased syncytiotrophoblast expression of leptin receptor ( p = 0.01) and SNAT-4 ( p < 0.001). Placental amino acid uptake was significantly stimulated by leptin in the lean group as compared to the obese group. Maternal weight gain and offspring birth weights were not different between groups. Conclusion Maternal obesity was accompanied by decreased placental SNAT activity associated with maternal hyperleptinemia and placental leptin resistance in spite of appropriate maternal weight gain and normally grown neonates. These findings suggest altered placental function that may have clinical implications in obese pregnant women.

Luminal leptin inhibits l-glutamine transport in rat small intestine: involvement of ASCT2 and B0AT1

L-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B(0)AT1/SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na(+)-dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B(0)AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na(+)-induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; I(C50) = approximately 0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B(0)AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B(0)AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B(0)AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression.

Tissue-Specific Amino Acid Transporter Partners ACE2 and Collectrin Differentially Interact With Hartnup Mutations

Background & AimsHartnup amino acid transporter B0AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B0AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2.MethodsBecause collectrin is almost absent from small intestine, we tested the hypothesis that it is ACE2 that interacts with B0AT1 in enterocytes. Furthermore, because B0AT1 expression depends on an associated protein, we tested the hypothesis that Hartnup-causing B0AT1 mutations differentially impact on B0AT1 interaction with intestinal and kidney accessory proteins.ResultsImmunofluorescence, coimmunoprecipitation, and functional experiments using wild-type and ace2-null mice showed that expression of B0AT1 in small intestine critically depends on ACE2. Coexpressing new and previously identified Hartnup disorder–causing missense mutations of B0AT1 with either collectrin or ACE2 in Xenopus laevis oocytes showed that the high-frequency D173N and the newly identified P265L mutant B0AT1 transporters can still be activated by ACE2 but not collectrin coexpression. In contrast, the human A69T and R240Q B0AT1 mutants cannot be activated by either of the associated proteins, although they function as wild-type B0AT1 when expressed alone.ConclusionsWe thus show that ACE2 is necessary for the expression of the Hartnup transporter in intestine and suggest that the differential functional association of mutant B0AT1 transporters with ACE2 and collectrin in intestine and kidney, respectively, participates in the phenotypic heterogeneity of human Hartnup disorder.

Despite Increased ATF4 Binding at the C/EBP-ATF Composite Site following Activation of the Unfolded Protein Response, System A Transporter 2 (SNAT2) Transcription Activity Is Repressed in HepG2 Cells

The activated amino acid response (AAR) and unfolded protein response (UPR) stress signaling pathways converge at the phosphorylation of translation initiation factor eIF2alpha. This eIF2alpha modification suppresses global protein synthesis but enhances translation of selected mRNAs such as that for activating transcription factor 4 (ATF4). An ATF4 target gene, SNAT2 (system A sodium-dependent neutral amino acid transporter 2), contains a C/EBP-ATF site that binds ATF4 and triggers increased transcription during the AAR. However, the present studies show that despite increased ATF4 binding to the SNAT2 gene during UPR activation in HepG2 human hepatoma cells, transcription activity was not enhanced. Hyperacetylation of histone H3 and recruitment of the general transcription factors at the HepG2 SNAT2 promoter occurred in response to the AAR but not the UPR. In contrast, the UPR did enhance transcription from a plasmid-based reporter gene driven by a SNAT2 genomic fragment containing the C/EBP-ATF site. Simultaneous activation of the AAR and the UPR pathways revealed that the UPR actually suppressed the increased SNAT2 transcription by the AAR pathway, demonstrating that the UPR pathway generates a repressive signal that acts downstream of ATF4 binding.

Gene expression profiling in brain following acute systemic hypertonicity: novel genes possibly involved in osmoadaptation

In brain osmoprotective genes known to be involved in cellular osmoadaptation to hypertonicity, as well as the related transcription factor tonicity-responsive enhancer binding protein (TonEBP) are only expressed in some cell subsets. In the search for other genes possibly involved in osmoadaptation of brain cells we have analyzed, through microarray, the transcriptional profile of forebrain from rats subjected to 45 min, 90 min, and 6 h systemic hypertonicity. Microarray data were validated by quantitative real-time PCR. Around 23 000 genes gave a reliable hybridization signal. The number of genes showing a higher expression increased from around 15 (45 min) up to nearly 200 (6 h). Among about 30 immediate early genes (IEGs) encoding transcription factors, only Atf3, Verge, and Klf4 showed a rapid increased expression. TonEBP-mRNA tissue level and TonEBP-mRNA labeling in neurons remained unchanged whereas TonEBP labeling was rapidly increased in neurons. Sodium-dependent neutral amino acid transporter-2 (SNAT2) encoded by gene Slc38a2 showed a delayed increased expression. The rapid tonicity-induced activation of Atf3, Verge, and Klf4 may regulate genes involved in osmoadaptation. Nfat5 encoding TonEBP is not an IEG and the early tonicity-induced expression of TonEBP in neurons may result from translational activation. Increased expression of sodium-dependent neutral amino-acid transporter 2 may lead to the cellular accumulation of amino acids for adaptation to hypertonicity.

Glutamine metabolism and signaling in the liver

Glutamine is the most abundant amino acid in the human body and can be synthesized by almost all tissues by the glutamine synthetase (GS)-catalyzed amidation of glutamate. Hepatocytes have access to extracellular glutamine by the concentrative uptake via members of the sodium-dependent neutral amino acid transport systems N and A. Hepatic glutamine metabolism in connection with urea synthesis is importantly involved in systemic ammonia detoxication and pH regulation due to the unique regulatory properties of the liver-type glutaminase, the acinar compartimentation of urea and glutamine synthesis, and a cycling of glutamine between periportal and perivenous hepatocytes. Upregulation of GS expression in hepatocellular carcinoma is related to growth advantage and an enhanced metastatic potential. Glutamine is a potent activator of signal transduction. Recent progress concerns the understanding of glutamine-induced hepatocyte swelling and the downstream activation of integrins, Src, and MAP-kinases in the regulation of autophagic proteolysis, canalicular bile acid excretion, glycogen and fatty acid synthesis, insulin signaling, and protection from apoptosis. Most recently the first primary GS defect leading to inherited glutamine deficiency with fatal outcome was described in human. This review summarizes recent progress in the understanding of glutamine metabolism and signal transduction, which provides further rationale for the use of glutamine as a therapeutic tool.

The role of the neutral amino acid transporter SNAT2 in cell volume regulation

Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.

Oligonucleotide microarray analysis of differential transporter regulation in the regenerating rat liver

The aim of this study was to investigate the regulation of hepatic transport systems during liver regeneration. A DNA oligonucleotide microarray was developed with probes for 400 transcripts. Data were confirmed using real-time PCR and on a functional level in the perfused rat liver. Liver homogenates were taken 3-48 h following 2/3-hepatectomy in rats and compared with sham-operated and non-operated controls. A more than two-fold increase or decrease of expression was obtained in 183 genes following partial hepatectomy and in 16 genes in sham-operated rats. A strong induction during liver regeneration was detected for the amino acid transporters LAT4, SN2 and sodium-dependent neutral amino acid transporter (ASCT)2, whereas amino acid transport system (ATA)2 and ATA3 expressions remained unchanged. The upregulation of ASCT2 may be responsible for the increase in sodium-dependent neutral amino acid influx important for liver cell proliferation. Expression of the osmolyte transporters Smit, TauT and Bgt1 was almost unchanged indicating that osmolytes are not involved in the cell volume increase during liver regeneration. The basolateral bile salt transporter Ntcp messenger RNA (mRNA) was significantly downregulated, whereas bile salt export pump (Bsep) and multidrug resistance protein (Mrp)2 expressions remained almost unchanged. An increased mRNA expression following partial hepatectomy was detected for organic anion transporting polypeptide (Oatp)5, Octn1, Octn2 and SGLT2. In contrast, Mrp6, Oatp 2, Oatp 3, Oatp 4 and Oatp 7 were downregulated. A five-fold upregulation at the protein level was shown for the Na(+)-K(+)-2Cl- cotransporter sodium-potassium-2-chloride cotransporter (NKCC1). The data show a differential regulation of hepatic transport systems during liver regeneration.

Impact of forskolin and amino acid depletion upon System A activity and SNAT expression in BeWo cells

Amino acid transport System A (SysA) plays an important role in mediating the transplacental transfer of neutral amino acids from mother to fetus. Given that prior work has demonstrated that SysA activity is regulated, both over gestation and in response to dietary restriction during pregnancy, we examined the response of SysA activity and sodium-dependent neutral amino acid transporter (SNAT; responsible for SysA activity) expression to cAMP analogues and amino acid deprivation in BeWo cells, an accepted model of placental syncytia. SysA activity was unaffected by forskolin, a cAMP agonist, at 48 and 72 h. Amino acid depletion was associated with an up-regulation of SysA activity, largely mediated through an enhancement of SNAT2 (Slc38a2) expression at both the protein and mRNA level. SNAT1 (Slc38a1) expression did not change in response to amino acid depletion, while SNAT4 (Slc38a4) could not be detected. In summary, SysA activity in BeWo cells responds to amino acid depletion through the differential regulation of SNAT subtypes.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B(0). We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G --> A and 0.001 for 718C --> T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Hypoxia differentially regulates nutrient transport in rat jejunum regardless of luminal nutrient present.

Aggressive enteral nutrition and poor intestinal perfusion are hypothesized to play an important pathogenic role in nonocclusive small bowel necrosis. This study tests the hypothesis that glucose and glutamine transport are differentially regulated during hypoxia regardless of the luminal nutrient present. Sprague-Dawley rats (247 +/- 3 g; n = 16) were randomized to receive 1 h of intestinal hypoxia or serve as normoxic controls. During this hour, jejunal loops were randomized to receive in situ perfusions of mannitol, glucose, or glutamine. When compared with normoxic groups, glucose but not glutamine transport was impaired (P < 0.001) during hypoxia. Messenger RNA abundance of the sodium glucose cotransporter sodium-dependent glucose cotransporter-1 (SGLT-1) and neutral basic amino acid transporter B(o) did not differ with hypoxia or nutrient perfused. Jejunal brush-border SGLT-1 abundance was decreased (P = 0.039) with hypoxia; however, total cellular SGLT-1 protein abundance did not differ among treatment groups. These data indicate that SGLT-1 activity is regulated during hypoxia at the posttranslational level. Additional information regarding the mechanisms regulating nutrient transport in the hypoperfused intestine is critical for optimizing the composition of enteral nutrient formulas.

The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors.

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.-L. Blond, D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel-Fernandez, B. Mandrand, F. Mallet, and F.-L. Cosset, J. Virol. 74:3321-3329, 2000) and that it can pseudotype human immunodeficiency virus cores (D. S. An, Y. Xie, and I. S. Y. Chen, J. Virol. 75:3488-3489, 2001). By using cell-cell fusion and pseudotype virion infection assays, we found that HERV-W Env efficiently uses both hASCT2 and the related transporter hASCT1 (gene name, SLC1A4) as receptors. In addition, although HERV-W Env mediates only slight syncytium formation or infection of mouse cells, it utilizes the mouse transporters mASCT1 and mASCT2 when their sites for N-linked glycosylation are eliminated by mutagenesis. Consistent with their role as a battlefield in host-virus coevolution, the viral recognition regions in ASCT1 and ASCT2 of humans and mice are highly divergent compared with other regions of these proteins, and their ratios of nonsynonymous to synonymous nucleotide sequence changes are extremely large. The recognition of ASCT1 and ASCT2 despite this divergence of their sequences strongly suggests that the use of both receptors has been highly advantageous for survival and evolution of the HERV-W family of retroviruses.

Truncated Forms of the Dual Function Human ASCT2 Neutral Amino Acid Transporter/Retroviral Receptor Are Translationally Initiated at Multiple Alternative CUG and GUG Codons

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.