We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.3%) and mouse (87.6%) counterparts. Confocal microscopic examination of porcine CAT-1-GFP-expressing HEK293 cells revealed that porcine CAT-1 localizes on the plasma membrane. Amino acid uptake studies in Xenopus oocytes injected with cRNA encoding this protein demonstrated transport properties consistent with system y(+). Radiation hybrid mapping data indicate that the porcine SLC7A1 maps to the distal end of the short arm of pig chromosome 11 (SSC11). This map location is consistent with the known conservation of genome organization between human and pig and provides further confirmation that we have characterized the porcine orthologue of the human SLC7A1.

Tumor cells overexpress amino acid transporters to meet the increased demand for amino acids. PQ loop repeat-containing (PQLC)2 is a cationic amino acid transporter that might be involved in cancer progression. Here, we show that upregulation of PQLC2 is critical to gastric cancer (GC) development in vitro and in vivo. Both PQLC2 mRNA and protein were overexpressed in GC tissues, especially of the diffuse type. Overexpression of PQLC2 promoted cell growth, anchorage independence, and tumor formation in nude mice. This was due to activation of MEK/ERK1/2 and PI3K/AKT signaling. Conversely, PQLC2 knockdown caused growth arrest and cell death of cancer cells and suppressed tumor growth in a mouse xenograft model. These results suggest that targeting PQLC2 is an effective strategy for GC treatment.

Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.

Amino acids are major primary metabolites. Their uptake, translocation, compartmentation, and re-mobilization require a diverse set of cellular transporters. Here, the broadly expressed gene product of CATIONIC AMINO ACID TRANSPORTER 9 (CAT9) was identified as mainly localized to vesicular membranes that are involved in vacuolar trafficking, including those of the trans-Golgi network. In order to probe whether and how these compartments are involved in amino acid homeostasis, a loss-of-function cat9-1 mutant and ectopic over-expressor plants were isolated. Under restricted nitrogen supply in soil, cat9-1 showed a chlorotic phenotype, which was reversed in the over-expressors. The total soluble amino acid pools were affected in the mutants, but this was only significant under poor nitrogen supply. Upon nitrogen starvation, the soluble amino acid leaf pools were lower in the over-expressor, compared with cat9-1. Over-expression generally affected total soluble amino acid concentrations, slightly delayed development, and finally improved the survival upon severe nitrogen starvation. The results potentially identify a novel function of vesicular amino acid transport mediated by CAT9 in the cellular nitrogen-dependent amino acid homeostasis.

Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer-related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference-mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1-GFP in a GFP expression-dependent manner were selected from established hybridoma clones. Novel anti-CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno-histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti-CAT1 mAbs exhibited internalization activity, antibody-dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW-C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA.

Blockade of the mineralocorticoid receptor (MCR) has been shown to improve endothelial function far beyond blood pressure control. In the current studies we have looked at the effect of MCR antagonists on cationic amino acid transporter-1 (CAT-1), a major modulator of endothelial nitric oxide (NO) generation. Using radio-labeled arginine, {[3H] l-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with either spironolactone or eplerenone with or without silencing of MCR. Western blotting for CAT-1, PKCα and their phosphorylated forms were performed. NO generation was measured by using Griess reaction assay. Both Spironolactone and eplerenone significantly increased endothelial arginine transport, an effect which was further augmented by co-incubation with aldosterone, and blunted by either silencing of MCR or co-administration of amiloride. Following MCR blockade, we identified two bands for CAT-1. The addition of tunicamycin (an inhibitor of protein glycosylation) or MCR silencing resulted in disappearance of the extra band and prevented the increase in arginine transport. Only spironolactone decreased CAT-1 phosphorylation through inhibition of PKCα (CAT-1 inhibitor). Subsequently, incubation with either MCR antagonists significantly augmented NO2/NO3 levels (stable NO metabolites) and this was attenuated by silencing of MCR or tunicamycin. GO 6076 (PKCα inhibitor) intensified the increase of NO metabolites only in eplerenone treated cells. In conclusion spironolactone and eplerenone augment arginine transport and NO generation through modulation of CAT-1 in endothelial cells. Both MCR antagonists activate CAT-1 by inducing its glycosylation while only spironolactone inhibits PKCα.

Arginine is an amino acid that serves as a substrate for the enzymes nitric oxide synthase (NOS) and arginase, leading to synthesis of NO and ornithine, respectively. As such, arginine has the potential to influence diverse fundamental processes in the lung.

Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome.

The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL), depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM) with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS) expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important therapeutic and prophylactic strategy to treat VL.

Metformin used as a first-line drug to treat Type 2 Diabetes Mellitus is transported via organic cation channels to soft tissues. Mutations in the SLC22A1 gene, such as Gly401Ser, Ser189Leu, and Arg206Cys, may affect the drug's therapeutic effect on these patients. This study aims at proposing a potential structural model for drug interactions with the hOCT1 transporter, as well as the impact of these mutations at both topological and electronic structure levels on the channel's surface, from a chemical point of view with, in addition to exploring the frequency distribution. To chemically understand metformin diffusion, we used an open model from the protein model database, with ID PM0080367, viewed through UCSF Chimera. The effect of the mutations was assessed using computational hybrid Quantum Mechanics/Molecular Mechanics, based on the Austin Model 1 semi-empirical method using Spartan 18' software. The results demonstrate coupling energy for metformin with amino acids F, W, H and Y, because of the interaction between the metformin dication and the electron cloud of π orbitals. The mutations analyzed showed changes in the chemical polarity and topology of the structure. The proposed diffusion model is a possible approach to the interaction mechanism between metformin and its transporter, as well as the impacts of variants, suggesting structural changes in the action of the drug. Metformin efficacy considerably varies from one patient to another; this may be largely attributed to the presence of mutations on the SLC22A1 gene. This study aims at proposing a potential structural model for metformin-hOCT1 (SLC22A1) transporter interaction, as well as the identification of the effect of mutations G401S (rs34130495), S189L (rs34104736), and R206C (616C > T) of the SLC22A1 gene at the topological and electronic structure levels on the channel surfaces, from a chemical viewpoint. Our results demonstrated that the coupling energies for metformin with aromatic amino acids F, W, H and Y, because of the interaction between the metformin dication and the electron cloud of π orbitals. Changes in the chemical environment's polarity and the structure's topology were reported in the mutations assessed. The diffusion model proposed is a potential approach for the mechanism of interaction of metformin with its transporter and the effects of variants on the efficacy of the drug in the treatment of type 2 diabetes. The assessment of the frequency of these mutations in a sample of Colombian type 2 diabetes patients suggests that different SLC22A1 gene variants might be involved in reduced OCT1 activity in the Colombian population since none of these mutations were detected.

Cationic amino acid transport activity in a canine lens epithelial cells (LEC) line was investigated. The transporter activity of arginine was 0.424 ± 0.047 nmol/mg protein min, while the presence of N-ethylmaleimide, an inhibitor of the canine cationic amino acid transporter (CAT), reduced transport activity by 30%. A full-length cDNA sequence of canine CAT1 was 2558 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence of canine CAT1 showed similarities of 92.1% and 88.6% to those of the human and mouse, respectively. Western blot analysis detected a band at 70 kDa in a membrane protein sample of LEC. RT-PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined.

Amino acid (AA) transporters (AAT) control AA cellular fluxes across membranes, contributing to maintain cellular homeostasis. In this study, we took advantage of rainbow trout metabolic feature, which highly relies on dietary AA, to explore the cellular and physiological consequences of unbalanced diets on AAT dysregulations with a particular focus on cationic AAs (CAA), frequently underrepresented in plant-based diets. Results evidenced that 24 different CAAT are expressed in various trout tissues, part of which being subjected to AA- and CAA-dependent regulations, with y+LAT2 exchanger being prone to the strongest dysregulations. Moreover, CAA were shown to control two major AA-dependent activation pathways (namely mTOR and GCN2) but at different strength according to the CAA considered. A new feed formulation strategy has been put forward to improve specifically the CAA supplemented absorption in fish together with their growth performance. Such "precision formulation" strategy reveals high potential for nutrition practices, especially in aquaculture.

ATB(0,+) (SLC6A14) is a transporter specific towards neutral and cationic amino acids, known to be up-regulated in malignant tumor cells. We cloned cDNA for rATB(0,+) and expressed it in HEK 293 cells. The ATB(0,+) over-expression correlated with increased l-leucine transport, stimulated by protein kinase C (PKC) activator and attenuated by PKC inhibitors. Transport stimulation was correlated with phosphorylation on serine moiety of the transporter and its augmented plasma membrane presence. Immunoprecipitation experiments demonstrated ATB(0,+) interaction with PKCα, but not with other classical or novel PKC isoforms. Immunocytochemistry experiments showed a transfer of PKCα to plasma membrane upon phorbol ester activation and co-localization with ATB(0,+). The observed regulation of ATB(0,+) by PKC correlates with high activity of both proteins reported for cancer cells.

Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT) is the rate-limiting step in nitric oxide (NO) synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS) expression was investigated in endotoxin-induced uveitis (EIU). Methods. EIU was induced in Lewis rats by lipopolysaccharide (LPS) injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB) binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate the in vivo findings. Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition. Conclusions. CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

The C9orf72 protein is required for normal lysosome function. In support of such functions, C9orf72 forms a heterotrimeric complex with SMCR8 and WDR41 that is recruited to lysosomes when amino acids are scarce. These properties raise questions about the identity of the lysosomal binding partner of the C9orf72 complex and the amino acid-sensing mechanism that regulates C9orf72 complex abundance on lysosomes. We now demonstrate that an interaction with the lysosomal cationic amino acid transporter PQLC2 mediates C9orf72 complex recruitment to lysosomes. This is achieved through an interaction between PQLC2 and WDR41. The interaction between PQLC2 and the C9orf72 complex is negatively regulated by arginine, lysine, and histidine, the amino acids that PQLC2 transports across the membrane of lysosomes. These results define a new role for PQLC2 in the regulated recruitment of the C9orf72 complex to lysosomes and reveal a novel mechanism that allows cells to sense and respond to changes in the availability of cationic amino acids within lysosomes.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+-independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are approximately 48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+-dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L-type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Heteromeric amino acid transporters (HATs) catalyze the transmembrane movement of amino acids, comprising two subunits, a heavy chain and a light chain, linked by a disulfide bridge. The b0,+AT (SLC7A9) is a representative light chain of HATs, forming heterodimer with rBAT, a heavy chain which mediates the membrane trafficking of b0,+AT. The b0,+AT-rBAT complex is an obligatory exchanger, which mediates the influx of cystine and cationic amino acids and the efflux of neutral amino acids in kidney and small intestine. Here, we report the cryo-EM structure of the human b0,+AT-rBAT complex alone and in complex with arginine substrate at resolution of 2.7 and 2.3 Å, respectively. The overall structure of b0,+AT-rBAT exists as a dimer of heterodimer consistent with the previous study. A ligand molecule is bound to the substrate binding pocket, near which an occluded pocket is identified, to which we found that it is important for substrate transport.