Cloning and characterization of a brain-specific cationic amino acid transporter.

rCAT3 (at ationic mino acid ransporter 3), a cDNA that encodes a novel member of the murine CAT family was isolated. The protein encoded by rCAT3 contained 619 amino acids, 53-58% of which were identical with those of the murine CAT family proteins previously described (mouse CAT1, CAT2a, CAT2b, and rat CAT1). Transient expression of rCAT3 and L-[14C]arginine incorporation experiments in COS7 cells verified a high affinity system y+ transporter activity of rCAT3. First, rCAT3-mediated L-[14C]arginine incorporation was time-dependent and saturable with half-saturation constant (Km) values of 103 ± 12 μM (mean ± S.E., n = 3). Second, the incorporation was specific for cationic amino acids as evidenced from the inhibition by L-arginine, L-lysine, and L-ornithine. Third, neither sodium nor chloride ions in the extracellular medium were required for the activity. Fourth, the incorporation was inhibited by high potassium-induced membrane depolarization. On Northern blot using RNAs from various rat tissues, the expression of rCAT3 mRNA was restricted to the brain. These results indicated a role of rCAT3 in the system y+ transporter activity in the nervous tissue.

Nitric oxide, a highly diffusible molecule involved in signal transduction in the brain, is formed from the terminal guanidino group of L-arginine by an enzyme NO synthetase (NOS) 1 (1). Of the two classes of NOS isoforms (constitutive and inducible), neuronal NOS belongs to the constitutive type, and it is generally accepted that calcium/calmodulin signaling is the primary mechanism that regulates its activity (1,2). Several lines of evidence have suggested that besides the regulation of NOS activity, NO synthesis is also regulated by availability of the substrate L-arginine (3) and that L-arginine is provided to NOS via transport from the extracellular space rather than from the intracellular metabolic pool. For example, N-methyl-D-aspartate-induced NO production in neuroblastoma cells depends on extracellular L-arginine (4). NO synthesis by a lipopolysaccharide-activated macrophage was also dependent on extracellular L-arginine and was inhibited by L-lysine or L-ornithine (5). Inhibitory effects of these cationic amino acids on NO synthesis were also observed in brain synaptosomes (6). Furthermore, exogenous L-arginine enhanced NO synthesis in brain slices (7,8).
The amino acids are incorporated from the exterior medium to the intracellular space via carrier proteins on the cell surface. Among the several types of amino acid transport systems distinguished by substrate specificity, cationic amino acids such as L-arginine, L-lysine, and L-ornithine share the same transporter with a sodium-independent activity, designated system y ϩ (9). Although the presence of system y ϩ activity in neuronal/glial cells has been demonstrated by functional studies (10,11), a molecular basis for the activity remains unclear.
At least two genes, mCAT1 (mouse cationic amino acid transporter 1) and mCAT2, have been identified and verified to encode a high affinity system y ϩ activity (K m ϭ ϳ100 M) in murine non-neuronal tissues. mCAT1, originally identified as a receptor for an ecotropic retrovirus (12), was later found to encode a system y ϩ activity by expression studies in Xenopus oocytes (13,14). mCAT1 is widely expressed in various tissues and cell lines and is believed to be a ubiquitous form of the CAT gene family (14). mCAT2 gives rise to two alternative splice forms, mCAT2a and mCAT2b. mCAT2b, originally identified as a T-cell early activated gene (15), was also found to encode a system y ϩ activity with properties similar to that encoded by mCAT1 (16). mCAT2b has been shown to be expressed in some types of cells including T cells and vascular smooth muscle cells (17). A comparative study on the distribution of mCAT2b, however, is lacking, and it is not known whether neuronal/glial cells express this transporter. mCAT2a does not encode system y ϩ but a low affinity, high capacity transporter (18). The expression of mCAT2a is restricted to liver (18).
By using an RNase protection assay, Stoll et al. (19) demonstrated a marked enrichment of the rat CAT1 (rCAT1) mRNA in brain capillary, suggesting a role of rCAT1 in amino acid transport by endothelial cells at the blood-brain barrier. The relatively low expression level of rCAT1 in the whole brain mRNA might imply the presence of a brain-specific, but as yet unknown transporter distinct from rCAT1. More recently, Wu et al. (20) examined the tissue distribution of rCAT1 mRNA and found a ubiquitous distribution of a 7.9-kilobase pair hybridization signal and a brain-specific 3.4-kilobase pair hybridization signal, the latter of which might represent a brainspecific homologue of rCAT1.
In light of the regulation of NO synthesis by L-arginine transport, we wished to identify the molecular basis for the system y ϩ activity in brain. The current study employed homology screening of a rat brain cDNA library and led to the isolation of a novel member of the CAT gene family, designated here as rCAT3.
* This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
cDNA Cloning-A cDNA library from adult Wistar rat brain poly(A) ϩ RNA was constructed in gt10 vector (Stratagene, La Jolla, CA) (22), and hybridization screening of the library (ϳ10 6 clones) was performed as described (23). A single positive clone (rCAT3) was extracted from the plaque, and the EcoRI fragment of the phage was subcloned into pBlueScript SKϩ (Stratagene) to give pBSKrCAT3. The nucleotide sequence of the pBSKrCAT3 insert was determined as described above.
Transient Expression of rCAT3 in COS7 Cells and L-[ 14 C]Arginine Incorporation Assay-The EcoRI fragment of rCAT3 was subcloned into the mammalian expression vector pME18sfϪ to give pMErCAT3. COS7 cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a CO 2 incubator. The cells were seeded on six-well plates (10 5 cells/well) and transfected with pMErCAT3 or with vector plasmid (for basal incorporations) using LipofectAMINE TM (Life Technologies, Inc.) according to the manufacturer's instructions. Two days later, they were subjected to L-[ 14 C]arginine incorporation assay. The cells were washed twice with HEPESbuffered saline (HBS) (150 mM NaCl, 10 mM HEPES, pH 7.5, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM KCl) and further incubated in HBS at 37°C for 10 min. The reaction was started by changing the media to HBS containing L-[ 14 C]arginine (11 GBq/mmol). The concentrations of L-[ 14 C]arginine that were applied and the reaction time used are indicated in the figure legends. The reaction was stopped by washing the cells three times with ice-cold HBS. The cells were then lysed in 0.2 N NaOH, 1% sodium dodecyl sulfate, and the radioactivity incorporated was determined by a solid scintillation counter. Incorporation assays with L-[ 3 H]lysine (3.55 TBq/mmol) and L-[ 14 C]ornithine (9.69 GBq/ mmol) were done in exactly the same way. All of the radioactive amino acids were obtained from Amersham. Protein concentration of the cell lysate was determined using a MicroBCA kit (Pierce). All of the unlabeled amino acids and the three NOS inhibitors (N G -monomethyl-Larginine (L-NMMA)), L-N 5 -(1-iminoethyl)ornithine (L-NIO)), N G -nitro-L-arginine methyl ester (L-NAME)) used for the inhibition experiments were obtained from Sigma.
Northern Blotting-The probe was prepared by labeling the EcoRI fragment of pBSKrCAT3 with [ 32 P]dCTP (110 TBq/mmol, Amersham) exactly as described above. A rat multiple tissue Northern blot membrane was obtained from Clontech (Palo Alto, CA). Blotting procedures were as described (23). The blot was visualized for radioactivity with a BAS2000 image analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Statistical Analysis-Where necessary, statistical analysis was done by analysis of variance. transmembrane domains; asterisk, a potential N-linked glycosylation site; double underline, a putative protein kinase C phosphorylation site; plus, a leucine zipper motif. B, schematic representation of the putative membrane topology of rCAT3 predicted by a hydrophobicity analysis. C, homology alignments of rCAT3 and other CAT family proteins. Residues conserved in all members are boxed. rCAT3 Is a Novel Member of the Murine CAT Family-cDNA prepared by reverse transcription from rat cerebellum RNA served as a template for PCR. An ϳ550-base pair product was obtained using a primer set derived from the 12th and 14th TMs of mCAT1 (12). The deduced amino acid sequence of this PCR product displayed ϳ70% identity with that of the corresponding region of mCAT1 and served as a probe for library screening. By hybridization screening of a rat brain cDNA library under high stringency conditions, one positive clone (rCAT3) was isolated.
The nucleotide and deduced amino acid sequences of the rCAT3 insert (Fig. 1A) indicated that it encoded a complete coding sequence of rCAT3. The 5Ј-proximal ATG triplet is followed by a 1857-base pair open reading frame encoding 619 amino acid residues with a calculated molecular mass of ϳ67 kilodaltons. The sequence around this ATG fits the consensus sequence for eukaryotic translation initiation sites (24). rCAT3 lacks a readable signal sequence in the N terminus (25). Fig. 1B depicts a putative membrane topology of rCAT3 predicted by a hydrophobicity analysis (GeneWorks TM , IntelliGenetics, Moun-tain View, CA). rCAT3 contains 14 putative TMs. One potential N-linked glycosylation site is located on an extracellular loop between the 5th and 6th TMs, and one potential protein kinase C phosphorylation site is located on an intracellular loop between the 10th and 11th TMs. Another structural feature of rCAT3 is a leucine zipper consensus sequence in the first TM (Fig. 1A). Originally identified as a functional unit required for dimerization of DNA-binding proteins (26), the leucine zipper motif has also been found in membrane proteins including voltage-gated potassium channels (27), glucose transporters (28), and some neurotransmitter transporters (29), where it may mediate subunit oligomerization.
The predicted amino acid sequence of rCAT3 had a 53-58% identity with those of other system y ϩ transporters previously reported (Fig. 1C, GeneWorks TM , IntelliGenetics). The leucine zipper motif is completely conserved in all members of the CAT family. Although individual sequences are variable, all of them have a potential N-glycosylation site and a potential protein kinase C phosphorylation site on the same extra-and intracellular loops, respectively. mCAT2a and mCAT2b are believed to be alternative splicing products of the same gene, and their sequences differ from each other only in a 41-amino acid stretch encoding the loop between the 8th and 9th TMs and a

FIG. 2. Kinetics of rCAT3-mediated L-[ 14 C]arginine incorporation in COS7 cells.
A, time course. COS7 cells transfected with pMEr-CAT3 or vector plasmid were incubated with L-[ 14 C]arginine (100 nM) for the time indicated, and the radioactivity incorporated was determined as described under "Experimental Procedures." The incorporation was normalized for the protein recovered from each well, and the rCAT3-mediated incorporation was defined as the difference between the values from pMErCAT3-and vector-transfected cells. B, saturation isotherms. COS7 cells transfected with pMErCAT3 or vector plasmid were incubated for 10 min with increasing concentrations of L-[ 14 C]arginine. To obtain concentrations higher than 100 nM, L-[ 14 C]arginine (100 nM) was diluted with unlabeled L-arginine, and the radioactivities recovered were normalized for the reduced specific activities. rCAT3mediated incorporation was determined as described above. Inset is an Eadie-Hofstee plot of the same data. The means Ϯ S.E. of triplicate determinations obtained in a single experiment are shown in both A and B. These experiments were replicated three times with similar results. part of the 9th TM (amino acids 358 -398 of mCAT2b). This divergent sequence has been shown to be responsible for the differential kinetics of L-arginine transport activity encoded by mCAT2a and mCAT2b (16). Within this stretch, the sequence of rCAT3 is homologous with those of the others in an order of rCAT1 Ͼ mCAT1 Ͼ mCAT2b Ͼ mCAT2a (GeneWorks TM , IntelliGenetics).
System y ϩ Transport Activity of rCAT3-L-Arginine transport activity of rCAT3 was tested by transient expression of the cDNA and L-[ 14 C]arginine incorporation assays in COS7 cells. In every experiment, one-half of the plates were transfected with an empty vector plasmid and assayed in parallel. Basal incorporations exhibited by vector-transfected cells were 40 -50% of those exhibited by pMErCAT3-transfected cells. rCAT3mediated incorporations were calculated as the differences be-tween the values from pMErCAT3-and vector-transfected cells.
rCAT3-mediated incorporation of L-[ 14 C]arginine (100 nM) was time-dependent and was close to linear in the first 10 min (Fig. 2A). Therefore, the reaction time was set to 10 min in the following experiments. The rCAT3-mediated incorporation of L-[ 14 C]arginine was dose-dependent and was saturated over the concentration of 0.5 mM (Fig. 2B) as expected for a carriermediated process. Eadie-Hofstee plot analysis (Fig. 2B, inset) of the saturation isotherms gave half-saturation constant (K m ) values of 103 Ϯ 12 M (mean Ϯ S.E., n ϭ 3). The K m values obtained were comparable with those of the L-arginine incorporation in Xenopus oocytes mediated by mCAT1 (14) or mCAT2b (16), both of which were verified to encode a high affinity system y ϩ transporter. They were also comparable with those for the system y ϩ transport activity determined in brain slices, cultured neurons, and glial cells (10,11). In addition, they were at least twice as low as those of the L-arginine incorporation in Xenopus oocytes mediated by mCAT2a (18), which encodes a liver-specific, low affinity transporter distinct from system y ϩ .
The substrate specificity was examined by testing the ability of unlabeled amino acids to inhibit the rCAT3-mediated L-[ 14 C]arginine incorporation. Of the 22 naturally occurring L-amino acids, only the 3 basic amino acids (L-arginine, Llysine, and L-ornithine) competed the incorporation (Fig. 3). Both of the two structural analogues of L-arginine tested (Darginine and L-citrulline) also caused a significant inhibition of the incorporation (Fig. 3). There was, however, a statistically significant difference between the effects of L and D isomers of arginine, suggesting a stereospecificity of the incorporation. To demonstrate that the inhibitory effects of the cationic amino acids observed were due to direct competition for rCAT3, we conducted incorporation experiments of radioactive L-lysine and L-ornithine (Fig. 4). In both cases, the rCAT3-mediated incorporations were dose-dependent, and Eadie-Hofstee plot analysis (not shown) gave K m values of 147 Ϯ 22 and 219 Ϯ 26 M (mean Ϯ S.E., n ϭ 3) for incorporations of L-[ 3 H]lysine and L-[ 14 C]ornithine, respectively. The specificity for cationic amino acids, together with the high affinity for L-arginine, indicated a system y ϩ transport activity of rCAT3.
Next, we examined the dependence of the rCAT3 activity on extracellular monovalent ions and membrane potentials by manipulating the ionic constitutions of the extracellular medium. Neither the isotonic substitution of Na ϩ with Li ϩ nor that of Cl Ϫ with CH 3 COO Ϫ caused any change in the rCAT3mediated L-[ 14 C]arginine incorporation (data not shown). In contrast, substitution of Na ϩ with K ϩ caused a dose-dependent inhibition of the rCAT3 activity (Fig. 5). These results are consistent with the functional properties of system y ϩ described previously, i.e. it is dependent neither on the extracellular Na ϩ nor on Cl Ϫ (9) and is inhibited by high K ϩ -induced membrane depolarization (9,30,31).
Inhibition of rCAT3-mediated L-[ 14 C]Arginine Transport by NOS Inhibitors-Currently available NOS inhibitors are structural analogues of L-arginine, and some of them also inhibit L-arginine transport (10,32). Therefore, we examined the ability of three drugs, L-NMMA, L-NIO and L-NAME, to inhibit rCAT3-mediated L-[ 14 C]arginine incorporation in COS7 cells (Fig. 6). When the transfected cells were exposed to L-[ 14 C]arginine (100 nM) in the presence or absence of the NOS inhibitors (1 mM), both L-NMMA and L-NIO caused a significant inhibition of the rCAT3-mediated L-[ 14 C]arginine incorporation, while L-NAME caused only a marginal effect. There was also a statistically significant difference between the effects of L-NMMA and L-NIO. Thus, the rank order of potency of the three drugs was L-NMMA Ͼ L-NIO Ͼ Ͼ L-NAME. Similar rank order of potency was observed for these drugs to inhibit system y ϩ L-arginine transport in endothelial cells (32) and in neuronal cells (10). These results gave further evidence for the system y ϩ transport activity of rCAT3 and support the notion that the transport of L-NAME is mediated by a system different from y ϩ (32).
Brain-specific Expression of rCAT3 mRNA-On Northern blot of poly(A) ϩ RNA from various rat tissues, a single band with a size of 3.3 kilobase pairs was detected in the brain only with a rCAT3 cDNA probe (Fig. 7), suggesting the brain-specific expression of rCAT3. Although direct evidence is lacking, it is very likely that the brain-specific 3.4-kilobase pair hybridization signal of rCAT1 detected by Wu et al. (20) was rCAT3.
Conclusion-We have isolated a cDNA clone (rCAT3) that encodes a novel member of the murine CAT family. Expression and functional characterization of the gene verified a high affinity, system y ϩ transporter activity of rCAT3. The expression of rCAT3 mRNA is restricted to the brain. The rCAT3 cDNA will be an essential tool to further clarify the molecular basis of the system y ϩ activity in the nervous tissues and that of the regulation of NO synthesis by L-arginine transport.