Sequence analysis and spatiotemporal developmental distribution of the Cat-1-type transporter slc7a1a in zebrafish (Danio rerio).

Cationic amino acid transporter 1 (Cat-1 alias Slc7a1) is a Na+-independent carrier system involved in transport and absorption of the cationic amino acids lysine, arginine, histidine, and ornithine and has also been shown to be indispensable in a large variety of biological processes. Starting from isolated full-length zebrafish (Danio rerio) cDNA for slc7a1a, we performed comparative and phylogenetic sequence analysis, investigated the conservation of the gene during vertebrate evolution, and defined tissue expression during zebrafish development. Whole mount in situ hybridization first detected slc7a1a transcripts in somites, eyes, and brain at 14 h post-fertilization (hpf) with additional expression in the distal nephron at 24 hpf and in branchial arches at 3 days post-fertilization (dpf), with significant increase by 5 dpf. Taken together, the expression analysis of the zebrafish Cat-1 system gene slc7a1a suggests a functional role(s) during the early development of the central nervous system, muscle, gills, and kidney. Graphical abstract.

Cytoplasmic R-peptide of murine leukemia virus envelope protein negatively regulates its interaction with the cell surface receptor.

Cytoplasmic tails of envelope (Env) glycoproteins of many retroviruses inhibit their membrane fusion activity. The cytoplasmic 16-amino acid peptide of ecotropic murine leukemia virus (E-MLV) Env protein, called the R-peptide, also inhibits the membrane fusion activity of the Env protein. However, the molecular mechanism of the inhibition has not been elucidated yet. In this study, we found that R-peptide-containing Env protein of E-MLV binds to the cell surface receptor cationic amino acid transporter-1 (CAT-1) with weaker affinity than R-peptide-truncated Env protein. Consistent with this result, R-peptide-containing Env protein had less efficient inhibition of E-MLV vector infection than R-peptide-truncated Env protein. R-peptide truncation has been reported to induce conformational change in the surface subunit of E-MLV Env protein that interacts with the receptor. Taken together, our findings indicate that R-peptide truncation induces conformational change in the receptor-binding domain of the E-MLV Env protein and facilitates the Env-receptor interaction.

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor.

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse (Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1.

Analysis of cationic amino acid transport activity in canine lens epithelial cells.

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.

Binding specificity of the L-arginine transport systems in mouse macrophages and human cells overexpressing the cationic amino acid transporter hCAT-1.

The uptake of L-arginine into mouse peritoneal macrophages can be inhibited by numerous amino acids and derivatives. Kinetic studies showed an almost entirely competitive inhibition for both cationic and neutral amino acids and derivatives suggesting that the comparison of their binding specificity by using a quantitative structure-activity relationship (QSAR) study is reasonable. The properties of the most efficient inhibitors were the following: the length of the aliphatic side chain, a general structural similarity to L-arginine (>0.79), cationic character, L-configuration, the presence of an alpha-amino group (with a mean pK(a) of 9.41), the van der Waals volume (mean 225 A(3)) and a low logP value (mean: -2.99). The significance of four other descriptors (neutral character, presence and the pK(a) of an alpha-carboxyl group, and the presence of a modified guanidino group) is much lower. Similar results were obtained for the hCAT-1 cell line, but the significance of the descriptors was slightly different. The L-configuration, van der Waals volume, the low logP value and the length of aliphatic side chain were the most significant, while the pK(a) value of the side chain (mean pK(a)=11.6) was found to be more important than that of the alpha-amino group. In addition, the general similarity to L-arginine, the presence of an amino group in the terminal position of the side chain (Orn, Lys) and the basic character were significant descriptors, while the significance of the acidity is negligibly low. As a final conclusion, the following descriptors were found to be important generally for the cationic transporters: the van der Waals volume, hydrophobicity (log P); L-configuration; the size of the side chain; the general similarity to L-arginine; the presence of an alpha-amino group; the general basicity of the molecule; the pK(a) values of the alpha-amino group (in macrophages) or that of the side chain (in CAT-1 cells). These descriptors can be regarded as the general structurally important binding characteristics of the cationic amino transporters.

Targeting and penetration of virus receptor bearing cells by nanoparticles coated with envelope proteins of Moloney murine leukemia virus.

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection.

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.

Intracellular accumulation of L-Arg, kinetics of transport, and potassium leak conductance in oocytes from Xenopus laevis expressing hCAT-1, hCAT-2A, and hCAT-2B.

Cationic amino acid transporters play an important role in the intracellular supply of L-Arg and the generation of nitric oxide. Since the transport of L-Arg is voltage-dependent, we aimed at determining the intracellular L-Arg concentration and describing the transport of L-Arg in terms of Michaelis-Menten kinetics, taking into account membrane voltage. The human isoforms of the cationic amino acid transporters, hCAT-1, hCAT-2A, and hCAT-2B, were expressed in oocytes from Xenopus laevis and studied with the voltage clamp technique and in tracer experiments. We found that L-Arg was concentrated intracellularly by all hCAT isoforms and that influx and efflux, in the steady state of exchange, were nearly mirror images. Conductance measurements at symmetric concentrations of L-Arg (inside/outside) allowed us to determine KM and Vmax. The empty transporter of hCAT-2B featured an unexpected potassium conductance, which was inhibited by L-Arg.

Transcriptional control of the arginine/lysine transporter, cat-1, by physiological stress.

Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.

Role of a conserved amino-terminal sequence in the ecotropic MLV receptor mCAT1.

The amino-terminus of mCAT1 and homologous proteins is predicted to form a positively charged, amphipathic alpha helix on the cytoplasmic side of the plasma membrane. Peptides with similar sequence motifs often provide membrane anchors, protein-protein interaction domains, or intracellular transport-targeting signals. Deleting most of the cytoplasmic N-terminal sequence of mCAT1 led to reduced expression on the cell surface and accumulation in the endoplasmic reticulum but did not abrogate receptor function. Surprisingly, when the N-terminal 36 or 18 amino acids of mCAT1 were fused to green fluorescent protein (gfp), gfp accumulated almost exclusively in mitochondria. Mitochondrial targeting depended on arginines at positions 15 and 16 and was inhibitable by downstream transmembrane sequences. Although the full-length mCAT1 was not detected in mitochondria, the mitochondrial-targeting property of the N-terminal sequence fused to gfp is conserved in orthologous and paralogous proteins that diverged approximately 80 million years ago, suggesting a conserved biological function. We propose that the conserved N-terminal motif of CAT proteins provides a regulatable signal for transport to, or retention in, different cell membrane compartments.

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells.

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca(2+)-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in l-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 micro M). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 micro M), which activates novel PKC isoforms, did not affect l-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 micro M; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4.

By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2.