Two Amino Acid Residues Determine the Low Substrate Affinity of Human Cationic Amino Acid Transporter-2A*
Alice Habermeier,
Sabine Wolf,
Ursula Martinë,
Petra Gräf and
Ellen I. Closs
From the
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany
Received for publication, October 7, 2002
, and in revised form, March 5, 2003.
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ABSTRACT
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Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low affinity of both the extracellular and intracellular substrate-binding sites. These residues are located in the fourth intracellular loop, suggesting that they are not part of the translocation pathway. Rather, they may be responsible for the low affinity conformation of the substrate-binding sites. The sensitivity to trans-stimulation is not determined by the same amino acid residues as the substrate affinity and must involve a more complex interaction between individual amino acid residues. In addition to the 42-amino acid domain, the adjacent transmembrane domain X seems to be involved in this function.
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INTRODUCTION
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The cationic amino acid transporter (CAT)1 family comprises four members: CAT-1, -2A, -2B, and -3 (for review, see Refs. 1, 2, 3). All CAT proteins mediate Na+-independent transport of cationic amino acids. However, they differ in their substrate affinity and sensitivity to trans-stimulation and to pH changes. The transport properties of the mouse and human CAT isoforms (mCAT and hCAT, respectively) have been characterized in greatest detail, e.g. in transport studies in Xenopus laevis oocytes, either using radiolabeled amino acids or by measuring amino acid-induced membrane currents by whole cell voltage clamping. The apparent Km values for cationic amino acids reported for mouse and human CAT-1, -2B, and -3 (0.10.4 mM) are characteristic of system y+ (4, 5, 6, 7, 8, 9). In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and also a greater maximal velocity (10, 11). The CAT proteins differ also in their sensitivity to trans-stimulation. The activities of CAT-1, -2B, and -3 are stimulated by physiological concentrations of substrate at the trans-side of the plasma membrane (0.11 mM), a characteristic also consistent with system y+. The most pronounced trans-stimulation has been observed for CAT-1. In contrast, transport mediated by CAT-2A is largely independent of the presence of substrate at the trans-side of the membrane. So far, only little information is available about the role of specific CAT protein amino acid residues in the recognition and translocation of cationic amino acids. Glu107 has been shown to be essential for the transport activity of mCAT-1 (12). Located in or adjacent to transmembrane domain (TM) III and conserved in all other known CAT isoforms, this Glu residue is likely to be part of the substrate translocation pathway. However, the amino acid residues that determine the particular transport properties of individual CAT isoforms, such as substrate affinity and sensitivity to trans-stimulation, have not been identified.
As evidenced from analyses of data bases of both the human and mouse genomes, CAT-2A and -2B are products of the same gene. Two alternative forms of the sixth coding exon give rise to the two splice variants, which differ only in a stretch of 42 amino acids (6, 8, 11). All known CAT proteins exhibit quite similar hydrophobicity plots, suggesting that their structures in the membrane are similar. They are integral membrane proteins with 1214 putative TMs and intracellular N and C termini. According to the model with 14 TMs, the region divergent between CAT-2A and -2B is located in the fourth intracellular loop and in part of the adjacent TM IX. Interestingly, in this region, the three isoforms exhibiting similar transport properties (CAT-1, -2B, and -3) also show the highest percentage of amino acid sequence identity (3). It is noteworthy that CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in 20 residues (within the stretch of 42 amino acids). Replacement of a 80-amino acid fragment containing the corresponding 42-amino acid domain of mCAT-1 with that of mCAT-2A or -2B and vice versa lead to chimeric proteins with transport properties of the donor of that domain (including the apparent affinity for L-arginine and sensitivity to trans-stimulation) (6). In this study, we aimed to identify the amino acid residues in the 42-amino acid domain of hCAT-2A that are responsible for its distinct transport properties.
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EXPERIMENTAL PROCEDURES
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Site-directed Mutagenesis and Generation of Chimeric cDNAsSite-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, Heidelberg, Germany) and the oligonucleotides listed in Table I. Silent in-frame BamHI and SalI sites were introduced into the coding regions of hCAT-1 and -2A. These, sites as well as a NcoI and a KpnI site (both conserved in hCAT-1 and -2A), were used to construct chimeric cDNAs between hCAT-2A and -1. The first letters of the restriction enzymes used are included in the name of each chimera, e.g. hCAT-2A/1.BK is a chimera that contains the backbone of hCAT-2A and the BamHI/KpnI fragment of hCAT-1.
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TABLE I
Oligonucleotides used for site-directed mutagenesis The sequence of each oligonucleotide pair is given in the sense orientation.
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Enhanced Green Fluorescent Protein (EGFP) Fusion ConstructsA construct encoding EGFP fused to the C terminus of hCAT-1 (hCAT-1/EGFP-pSP64T) has been described previously (13). A construct encoding a fusion protein between hCAT-2A and EGFP (hCAT-2A/EGFP-pSP64T) was obtained as described previously for hCAT-2B (13).
Expression of cRNAs in X. laevis OocytesAll cDNAs were inserted into the BglII site of pSP64T (14). The plasmids were linearized, and cRNA was prepared by in vitro transcription from the SP6 promoter (mMessage mMachine in vitro transcription kit, Ambion, AMS Biotechnology Europe, Cambridgeshire, UK). 36 ng of cRNA (in 36 nl of H2O) were injected into each X. laevis oocyte (Dumont stages V and VI). Oocytes injected with 36 nl of water were used as controls.
Transport Studies in X. laevis OocytesL-Arginine uptake was determined 2 days after injection of cRNA as previously described (8). Briefly, oocytes were equilibrated for 2 h at 18 °C in uptake solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 5 mM Tris, pH 7.5) containing the indicated concentrations of unlabeled L-amino acids. The oocytes were then transferred to the same solution supplemented with L-[3H]arginine (510 µCi/ml; ICN, Eschwege, Germany). After a 15-min incubation (or 16-h incubation for steady-state experiments) at 20 °C, the oocytes were washed four times with ice-cold uptake solution and solubilized individually in 2% SDS. The incorporated radioactivity was determined in a liquid scintillation counter. For trans-stimulation experiments, cRNA-injected oocytes were each injected a second time with 3.6 nmol of L-[3H]arginine (3.6 nCi) in 36 nl of water. The oocytes were then immediately transferred into uptake solution containing either 1 mM L-arginine or no cationic amino acids. After a 30-min incubation at 20 °C, the L-[3H]arginine that had accumulated in the uptake solution was determined by liquid scintillation counting.
Generation of an Immune Plasma Specific for hCAT-2A cDNA fragment coding for the 57 C-terminal amino acids of hCAT-2 was cloned in-frame into pATH-1,3' to the coding region of tryptophan E (15). The resulting plasmid was transfected into Escherichia coli XL-1 Blue (Stratagene). Expression of the tryptophan E/hCAT-2 fusion protein was induced by growth in tryptophan-free M9 medium containing 10 µg/ml 3-
-indoleacrylic acid (Sigma, Deisenhofen, Germany) for 4 h at 37 °C. Bacteria were lysed by sonication in buffer composed of 100 mM KCl, 25 mM HEPES, pH 7.6, 0.1 mM EDTA, 12 mM MgCl2, 10% glycerol, and 0.1% Nonidet P-40 containing 1 mM dithiothreitol, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 mM NaHSO3, and 0.5 mg/ml lysozyme. The lysates were spun at 26,000 x g, and the supernatants (
15 mg of protein) were separated by 12.5% SDS-PAGE. After Coomassie Blue staining, the gel portion containing the fusion protein was cut out and homogenized in 1 ml of H2O/g of gel using a 26-gauge needle. 6-week-old rabbits were immunized with 300 µl of homogenate (250 µg of fusion protein) and an equal volume of complete Freund's adjuvant (Invitrogen, Eggenstein, Germany). The immune plasma was collected after boosting the rabbits three times (every 3 weeks) with 250 µg of fusion protein (in 300 µl) and 1 volume of incomplete Freund's adjuvant.
Affinity Purification of Immune Plasma for hCAT-2A cDNA fragment coding for the 57 C-terminal amino acids of hCAT-2 was cloned in-frame into pGEX-3X 3' to the coding region of glutathione S-transferase (GST) (16). The resulting plasmid was transfected into E. coli XL-1 Blue. Expression of the GST/hCAT-2 fusion protein was induced by growth in LB medium containing 0.1 mM isopropyl-
-D-thiogalactopyranoside (Roche Applied Science, Mannheim, Germany) for 5 h at 37 °C. Bacteria were lysed on ice by sonication in phosphate-buffered saline (PBS; 2.7 mM KCl, 140 mM NaCl, 1.8 mM KH2PO4, and 10 mM Na2HPO4). After addition of 0.1% Triton X-100, the lysates were spun at 7500 x g, and the fusion protein in the supernatants was purified using S-glutathione-Sepharose (17). Immune plasma (2 ml) was heat-inactivated for 30 min at 56 °C, diluted 1:1 with PBS, and applied to a Poly-Prep chromatography column (Bio-Rad, Munich, Germany) containing 8 mg of GST/hCAT-2 fusion protein coupled to 4 ml of Affi-Gel 10 (Bio-Rad) at 4 °C for 15 h. After washing the column with 12 ml of PBS, antibodies were eluted with 0.1 M glycine HCl, pH 2.5, and 1 M NaCl; neutralized with 0.1 volume of 1 M Tris-HCl, pH 8; and dialyzed against PBS at 4 °C for 15 h.
Western BlotsOocytes were lysed by vortexing in radioimmune precipitation assay buffer (1% deoxycholate, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM MgCl2, 10 mM Tris-HCl, pH 7.2, and 1 mM phenylmethylsulfonyl fluoride) 2 days after injection with wild-type, mutant, or chimeric hCAT-2A cRNA or with water (five oocytes/25 µl of buffer). Lysates were then treated with N-glycosidase F (4 units/25 µl; Roche Applied Science) for 1 h at 37 °C. Radioimmune precipitation assay buffer (75 µl) containing 8 M urea was then added. The samples were spun at 14,000 x g. After determining the protein concentration of the supernatant (using the Bradford reaction, Bio-Rad), an equal volume of sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 5% SDS, 2% 2-mercaptoethanol, 0.001% bromphenol blue, and 1 mM phenylmethylsulfonyl fluoride) was added.
Lysates (20 µg of protein) were separated by 10% SDS-PAGE and then blotted onto nitrocellulose membranes (Protran 83, Schleicher & Schüll, Dassel, Germany). Staining for hCAT-2 proteins was achieved by sequential incubations in Blotto (50 mM Tris-HCl, pH 8, 2 mM CaCl2, 0.01% antifoam A (Sigma), 0.05% Tween 20, and 5% nonfat dry milk) containing 10% goat serum for 2 h at room temperature; a 1:100 dilution of anti-hCAT-2 polyclonal antibody in PBS containing 1% bovine serum albumin and 0.1% Tween 20 overnight at 4 °C; three times in Blotto for 15 min at room temperature; a 1:10,000 dilution of peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Calbiochem, Bad Soden, Germany) in Blotto for1hat room temperature; three times in 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20; once in 10 mM Tris-HCl, pH 8, and 150 mM NaCl; and finally in chemiluminescence reagent (Renaissance, PerkinElmer Life Sciences, Bad Homburg, Germany) for 1 min. The membranes were then immediately exposed to x-ray films (Agfa, Leverkusen, Germany). For each experiment, two to four different exposure times were used for quantification. Rabbit anti-GFP peptide polyclonal antibodies (Clontech, Heidelberg, Germany) were used as primary antibodies (1:500) for the detection of EGFP fusion proteins. For standardization, membranes were stripped with 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol for 30 min at 50 °C and stained with anti-
-tubulin monoclonal antibody (1:1000; Sigma) and peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:3000; Sigma).
Biotinylation of Cell Surface ProteinsOocytes were rinsed with ice-cold modified PBS (1.76 mM KH2PO4, 2 mM KCl, 10.1 mM Na2HPO4, and 0.1 M NaCl,) containing 0.1 mM CaCl2 and 1 mM MgCl2 and incubated in this same solution supplemented with 1 mg/ml sulfosuccinimidobiotin (EZ-LinkTM sulfosuccinimidyl-2-(biotinamido)ethyl 1,3'-dithiopropionate, Pierce) for 30 min at room temperature. Oocytes were then rinsed four times with modified calcium/magnesium/PBS containing 50 mM NH4Cl and incubated in this buffer for 10 min at 4 °C to quench the unreacted biotin. Oocytes were lysed in radioimmune precipitation assay buffer containing protease inhibitors. After removal of the cell debris by centrifugation, biotinylated proteins were batch-extracted using avidin-coated Sepharose beads (immobilized NeutrAvidinTM, Pierce). Biotinylated proteins were released from the beads by incubation in sample buffer containing 8 M urea for 10 min at 37 °C and analyzed by Western blotting.
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RESULTS
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Apparent Substrate AffinityTo elucidate which amino acid residues are responsible for the distinct transport properties of hCAT-2A, we first replaced peptide fragments of different lengths in hCAT-2A with the corresponding fragments in hCAT-1 (Fig. 1). To this end, we introduced a BamHI and a SalI recognition site in the hCAT-1 and -2A cDNAs without changing the reading frame. We used these sites as well as the conserved NcoI and KpnI sites to exchange fragments of 80 (BamHI/KpnI), 57 (BamHI/SalI), and 46 (NcoI/SalI) amino acids, resulting in the chimeras hCAT-2A/1.BK, hCAT-2A/1.BS, and hCAT-2A/1.NS, respectively. The 80- and 57-amino acid fragments contain the entire region that is divergent between hCAT-2A and -2B, whereas the 46-amino acid fragment lacks the first nine amino acids of the divergent region. The apparent substrate affinity of the chimeras was determined by expression in X. laevis oocytes and by measurement of the uptake of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 mM L-[3H]arginine over 15 min. The apparent half-saturating L-arginine concentrations (Km) were determined by fitting the data according to the Eadie-Hofstee equation after subtraction of the values obtained with water-injected oocytes. All three chimeras exhibited Km values significantly smaller than the Km of hCAT-2A and statistically not different from the Km of hCAT-1 (Fig. 2).

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FIG. 1. Models of hCAT-2A and the chimeric and mutant proteins. A, model of hCAT-2A with 14 putative TMs (numbered IXIV) predicted by the TopPred 2 program (18). The amino acids in the protein region where hCAT-2A differs from hCAT-2B are indicated as closed circles. The positions corresponding to the restriction enzyme recognition sites in the cDNA of hCAT-2A that were used to create chimeric proteins are boxed. These were either already present (NcoI and KpnI sites) or introduced using site-directed mutagenesis (BamHI and SalI sites). The arrows indicate the two point mutations introduced into hCAT-2A. The branched lines indicate putative glycosylation sites. B, sequence comparison of hCAT-1, -2A, -2B, and -3 in the area where hCAT-2A differs from hCAT-2B. Amino acid sequences were aligned using the BLASTp program. The small horizontal brackets mark the positions of restriction enzyme recognition sites in the corresponding cDNAs of hCAT-1 and -2A that were used to create chimeric proteins. Numbers on top refer to the positions of the respective amino acid residues in hCAT-2A. The 42-amino acid domain that differs between hCAT-2A and -2B is marked by vertical brackets. The large horizontal brackets indicate TMs predicted by the TopPred 2 program. The arrows indicate the two most striking differences in the sequence of low affinity hCAT-2A compared with the sequences of high affinity hCAT-1, -2B, and -3. C, schemes of the chimeric and mutant hCAT-2A proteins. The white boxes indicate the hCAT-1 sequence; the gray boxes indicate the sequence common in hCAT-2A and -2B; and the black boxes indicate sequence specific for hCAT-2A.
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FIG. 2. Apparent Km values for L-arginine of wild-type, chimeric, and mutant hCAT-2A proteins in comparison with those of hCAT-1. X. laevis oocytes were injected with 36 ng of cRNA (in 36 nl of water) encoding the respective wild-type, chimeric, or mutant hCAT-2A or with 36 nl of water alone. 2 days later, uptake of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 mM L-[3H]arginine was measured over 15 min at 20 °C. The apparent Km values were determined using the Eadie-Hofstee equation (after subtraction of the values obtained with water-injected oocytes). Bars represent means ± S.E. (n = 48), with four to six replicates each. Statistical analysis was performed using analysis of variance with Bonferroni's post-hoc test. ***, **, and *, p < 0.001, 0.01, and 0.05, respectively; ns (not significant), p > 0.05.
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Comparison of the CAT proteins in the exchanged region revealed two striking differences in the amino acid sequences of hCAT-2A and the high affinity CAT isoforms: an arginine residue (carrying a positive charge) at position 369 of hCAT-2A, where the high affinity isoforms have a negatively charged glutamic acid; and a missing amino acid residue at position 381 of hCAT-2A, where the high affinity isoforms contain an asparagine or histidine residue (Fig. 1B). Therefore, Arg369 in hCAT-2A was mutated to Glu, and Asn was inserted in position 381 (hCAT-2A(R369E/N381i)). Transport studies in X. laevis oocytes revealed Km values intermediate between those of hCAT-2A and -1 for the hCAT-2A proteins carrying either single mutation (Fig. 2). The hCAT-2A double mutant exhibited Km values indistinguishable from those of hCAT-1.
In a previous study, we established that, also at the inner side of the membrane, hCAT-2A exhibits a significantly lower apparent substrate affinity than hCAT-1 (8). At saturating extracellular substrate concentrations (Vmax for influx), an apparent steady state is reached when the intracellular substrate-binding sites are also saturated (Vmax for efflux). Cells will therefore accumulate more substrate when expressing a transporter with low substrate affinity at the cytoplasmic side compared with a transporter with high substrate affinity at the cytoplasmic side. The apparent steady-state accumulation under saturating extracellular substrate concentrations thus represents an indirect measurement of the substrate affinity at the intracellular side. To this end, we measured the accumulation of tritiated L-arginine over 6 h in oocytes incubated in isotonic salt solution containing 10 mM L-[3H]arginine (Fig. 3). As observed previously (8), hCAT-2A-expressing oocytes accumulated considerably more L-arginine over the 6-h incubation time than hCAT-1-expressing oocytes (7.2 ± 0.27 versus 1.7 ± 0.13 nmol/oocyte, n = 45). Oocytes expressing the double mutant hCAT-2A(R369E/N381i) accumulated also significantly less L-arginine (3.8 ± 0.23 nmol/oocyte, n = 45) than oocytes expressing hCAT-2A.
Maximal Transport ActivityWe routinely observe a higher maximal transport activity in oocytes expressing hCAT-2A compared with those expressing hCAT-1 (5.1 ± 0.88 versus 1.8 ± 0.19 nmol/oocyte/h; p < 0.001). However, it had not been elucidated if this difference between the two proteins is due to differences in the specific activity or rather in the expression level in the plasma membrane. To address this point, we fused EGFP to the C terminus of hCAT-1 and -2A and determined the transport activities and expression levels of the two proteins in X. laevis oocytes. Both proteins were predominantly localized to the plasma membrane (Fig. 4, A and B). However, compared with hCAT-1/EGFP, hCAT-2A/EGFP demonstrated a 3-fold higher maximal transport activity (determined at 10 mM L-arginine) in relation to its total and cell surface protein expression (determined in Western blot analyses using anti-GFP antibody) (Fig. 4C). These data indicate that hCAT-2A has indeed a higher maximal transport activity compared with hCAT-1.

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FIG. 4. Comparison of the maximal transport activities of hCAT-1/EGFP and hCAT-2A/EGFP. X. laevis oocytes were injected with cRNA encoding hCAT-1 or hCAT-2A with EGFP fused to the C terminus (or with water alone) and analyzed 2 days later. A and B, shown are fluorescent micrographs of cryostat sections (12 µm) from oocytes expressing hCAT-1/EGFP and hCAT-2A/EGFP, respectively. C, the uptake of 10 mM L-[3H]arginine over 15 min at 20 °C was measured, and the values obtained with water-injected oocytes were subtracted. To determine the cell surface expression of the transporters, oocytes were exposed to biotin prior to lysis, and the biotinylated proteins were isolated with streptavidin. The total and cell surface protein expression of each transporter were quantified by Western blotting using a commercial anti-GFP-antibody. The values for L-arginine transport were then divided by the respective protein values and expressed as the percentage of the values obtained for hCAT-1 (100%). Bars represent means ± S.E. (n = 35).
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We then wondered whether the same protein domain that determines the apparent substrate affinity of hCAT-2A also determines its maximal transport activity. To quantify the protein expression levels of wild-type and mutant hCAT-2A proteins, we generated immune plasma against the C terminus of hCAT-2. This plasma recognized a protein band of
70 kDa in N-glycosidase-treated lysates from X. laevis oocytes expressing wild-type or any mutant hCAT-2A, but not in lysates from control oocytes (Fig. 5A). (The calculated molecular mass of hCAT-2A is 72 kDa.) When the lysates were not treated with N-glycosidase, the antibody stained a broad protein band of 100150 kDa (Fig. 5B). The maximal transport activities of wild-type and mutant hCAT-2A proteins were calculated from the respective Eadie-Hofstee plots described above. Only oocytes expressing the single mutant hCAT-2A(N381i) and the double mutant hCAT-2A(R369E/N381i) had significantly reduced transport activities compared with oocytes expressing hCAT-2A (Fig. 6A). The specific activity of each transporter was then calculated by dividing the Vmax values by the respective protein values obtained by densitometry of several Western blots (Fig. 6, B and C). The chimeras hCAT-2/1.BS and hCAT-2/1.NS showed specific activities indistinguishable from that of hCAT-2A. This indicates that the hCAT-1 fragment introduced into the hCAT-2 backbone does not determine the specific activity. Also, the specific activity of the single mutant hCAT-2A(R369E) was equal to that of hCAT-2A. The chimera hCAT-2/1.BK and the single mutant hCAT-2A(N381i) had slightly reduced specific activities. A larger reduction was observed for the double mutant.

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FIG. 5. Protein expression of wild-type, chimeric, and mutant hCAT-2A proteins. Western blotting was performed with lysates from oocytes prepared 2 days after injection of cRNA from one of the indicated transporters or of water alone. Protein (20 µg/lane) was separated by 10% SDS-PAGE and blotted onto a membrane, and the membrane was incubated with affinity-purified anti-hCAT-1 antibody (A and B, upper panels). Lysates in A were treated with N-glycosidase F. Black and white arrows indicate the glycosylated and deglycosylated hCAT-2A proteins, respectively. The blots were then stripped and incubated with anti- -tubulin monoclonal antibody for standardization (A and B, lower panels).
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FIG. 6. Assessment of the specific transport activities of wild-type, chimeric, and mutant hCAT-2A proteins. A, the maximal transport activity of each transporter was calculated from the experiments described in the legend to Fig. 2. Bars represent means ± S.E. (n = 48), with four to six replicates each. B, protein expression was quantified by densitometry of several Western blots. For standardization, values obtained for each transporter were divided by the respective values for tubulin. The values obtained for hCAT-2A were set as 100% in each gel. Bars represent means ± S.E. (n = 3), with two to three replicates each. C, the Vmax values in A were divided by the respective protein values in B and expressed as the percentage of the values obtained for hCAT-2A (100%).
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Sensitivity to trans-Stimulationtrans-Stimulation is defined as the stimulation of transport by substrate at the trans-side of the membrane. The sensitivity of the chimeric and mutant hCAT-2A proteins to trans-stimulation was examined in efflux experiments. To this end, L-arginine efflux from oocytes injected with L-[3H]arginine was measured in isotonic salt solution containing either 1 mM unlabeled L-arginine (trans-substrate) or no trans-substrate. As observed previously (8), hCAT-2A-mediated efflux was not influenced by the presence of trans-substrate. In contrast, efflux mediated by hCAT-1 and the chimeras was significantly reduced in the absence of trans-substrate compared with 1 mM trans-substrate (Fig. 7A). The hCAT-2A(R369E) mutant had an intermediate phenotype. Surprisingly, neither the hCAT-2A(N381i) mutant nor the double mutant was sensitive to trans-stimulation. Because the two chimeras containing the SalI/KpnI fragment of hCAT-2A (hCAT-2/1.BS and hCAT-2A/1.NS) (Fig. 7A) seemed to be less sensitive to trans-stimulation than hCAT-2/1.BK, we wondered whether the SalI/KpnI fragment of hCAT-1 influences also the sensitivity to trans-stimulation. To address this question, we constructed new chimeras between hCAT-2A and -1 (Fig. 7B). In fact, the hCAT-2A/1.SK chimera, carrying only the SalI/KpnI fragment of hCAT-1, was sensitive to trans-stimulation with a phenotype intermediate between those of hCAT-1 and -2A and similar to that of the hCAT-2A(R369E) mutant (Fig. 7C). However, the hCAT-2A/1.SK chimera carrying, in addition, the R369E mutation was not more sensitive to trans-stimulation than either hCAT-2A/1.SK or hCAT-2A(R369E) alone. The hCAT-2A/1.NK chimera showed a similar reduction of transport activity in the absence of trans-substrate compared with hCAT-2A/1.BK. This demonstrates that the short BamHI/NcoI fragment does not contribute to trans-stimulation.

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FIG. 7. trans-Stimulation of wild-type, chimeric, and mutant hCAT-2A proteins in comparison with hCAT-1. A, trans-stimulation was examined in efflux experiments. Each transporter was expressed in X. laevis oocytes as described in the legend to Fig. 2. 2 days after injection of cRNA, oocytes were injected with 36 nl of L-[3H]arginine (3.6 nCi, 3.6 nmol). L-Arginine efflux was measured by transferring oocytes (three oocytes/200 µl) immediately into isotonic salt solution containing either 1 mM unlabeled L-arginine (trans-substrate) or no trans-substrate. The amount of L-[3H]arginine (disintegrations/min) in the incubation solution accumulated over 30 min was determined, and the values obtained with water-injected oocytes were subtracted. Efflux rates with no trans-substrate were calculated as percent of the mean of the efflux observed at 1 mM trans-substrate. Bars represent means ± S.E. (n = 48), with three to six replicates each. Statistical analysis was performed using analysis of variance with Bonferroni's post-hoc test. ***, **, and *, p < 0.001, 0.01, and 0.05, respectively; ns (not significant), p > 0.05. B, shown are schemes of the additional chimeras examined in efflux experiments. The hCAT-2A backbone has been truncated as indicated by the slanted lines. C, trans-stimulation experiments with the new chimeras were performed as described for A.
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Interestingly, the maximal efflux measured at 1 mM trans-substrate was about three to four times higher in hCAT-2A-expressing oocytes than in hCAT-1-expressing oocytes (Table II). All chimeras had similar transport activities compared with hCAT-2A with the exception of hCAT-2A/1.SK, which exhibited a significantly reduced efflux rate. Other than in the influx experiments, all of the mutant hCAT-2A proteins showed reduced activities in the efflux assay. The lowest activity was observed for hCAT-2A(R369E).
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TABLE II
L-[3H] Arginine efflux at 1 mM extracellular L-arginine Shown are the means ± S.E. (n) obtained in the efflux experiments described in the legend to Fig. 6 after subtracting the values obtained with water-injected oocytes (766 ± 91 dpm).
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DISCUSSION
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Our data demonstrate that a fragment of 80 amino acids extending from the end of TM VIII to the end of TM X (Fig. 1A) can confer the transport properties of hCAT-1 to hCAT-2A. This fragment contains the protein domain of 42 amino acids corresponding to the domain divergent between hCAT-2A and -2B (comprising the fourth intracellular loop and part of TM IX) (Fig. 1, A and B). The reciprocal chimera (hCAT-1/2A.BK) carrying the 80 amino acids from hCAT-2A in the hCAT-1 backbone had the transport properties of hCAT-2A (data not shown). This is in agreement with previous results from mCAT proteins (6). Also smaller fragments containing only 57 or 46 amino acids of hCAT-1 could transfer the high substrate affinity and sensitivity to trans-stimulation of hCAT-1 to hCAT-2A. Interestingly, the reciprocal chimeras had very low transport activities (data not shown), suggesting important intramolecular interactions within the 80-amino acid fragment. The two hCAT-1 chimeras carrying 57 and 80 amino acids of hCAT-2A, respectively, differ only in three amino acid residues located in TM X (Fig. 1A). These sequence changes in TM X of hCAT-2A or -2B versus hCAT-1 are conservative (Fig. 1B). However, the particular residues are conserved in the respective orthologs of different mammalian species, suggesting that they might be crucial for the transport function. This is also demonstrated by the reduced efflux activity of the hCAT-2A/1.SK chimera, which differs from hCAT-2A only in these three residues (Table II).
Mutation of two amino acid residues (Arg369 to Glu and insertion of Asn381) was sufficient to decrease the apparent Km of hCAT-2A at the extracellular face to values similar to those of hCAT-1. The localization of the two residues in an intracellular loop (as opposed to a TM) suggests that they are not part of the substrate-binding site, but influence substrate binding indirectly. The reciprocal mutant of hCAT-1 (hCAT-1(E367R/N379d) had no transport activity (data not shown), demonstrating that the two amino acid residues (Glu367 and Asn379) are essential for transport function in the hCAT-1 backbone. It seems that CAT-2 is more flexible in accepting changes in the amino acid sequence, probably because it can naturally accommodate both a low and a high affinity domain. Our steady-state experiments revealed that the two mutations in hCAT-2A also increased the apparent affinity of the intracellular substrate-binding site. Using high pressure liquid chromatographic analyses, we have previously established that hCAT-2A-expressing oocytes exhibit higher L-arginine levels than hCAT-1-expressing oocytes when incubated in high extracellular L-arginine concentrations (8). Therefore, the larger accumulation of tritiated L-arginine in hCAT-2A-expressing oocytes versus hCAT-1-expressing and hCAT-2A(R369E/N381i)-expressing oocytes observed here indeed reflects higher intracellular L-arginine levels that must be reached before efflux can occur at a maximal rate. The simultaneous change in the affinity of the intracellular and extracellular substrate-binding sites further supports the concept of an indirect influence of the two residues on substrate binding.
Using fusion proteins between hCAT-1 or hCAT-2A and EGFP, we demonstrated that the two CAT isoforms differ indeed in their maximal transport activities. The ratio of transport activity to protein expression stayed about the same irrespective of whether the total protein expressed or only the protein expressed at the cell surface was taken into consideration. The EGFP fusion partner did not seem to influence the transport properties of the hCAT proteins, as the Km and Vmax values obtained for the hCAT fusion proteins were not different from those determined for the native hCAT isoforms (Ref. 13 and data not shown). In influx and efflux experiments, all hCAT-2A/1 chimeras had maximal activities similar to those of hCAT-2A. Therefore, the specific transport activities of the hCAT proteins do not seem to be controlled by the same protein domain that determines the apparent substrate affinity and the sensitivity to trans-stimulation. Our results with the hCAT chimeras are in contrast to our earlier study carried out with chimeras of mCAT-1, -2A, and -2B, where the Vmax values obtained for each chimera were very similar to the Vmax values obtained for the respective donor of the 80-amino acid fragment (6). However, in these studies, we did not control for protein expression and therefore could not determine the specific activity of each transporter. The reduced transport activity of the double mutant hCAT-2A(R369E/N381i) in the influx experiments likely resulted from an interference with the overall protein structure, e.g. the reciprocal mutant had no activity. Interestingly, in efflux experiments, the activity of the double mutant was higher than that of hCAT-2A(R369E), whereas the opposite was observed in influx experiments. This demonstrates that the two transport pathways are not necessarily coupled.
The lack of trans-stimulation indicates that a transporter can move between the outward and inward facing conformation without substrate, therefore mediating net transport. In contrast, a strongly trans-stimulated transporter works only in the exchange mode. In our previous work, we extensively characterized the trans-stimulation of CAT-1 and -2A, demonstrating that both transporters work symmetrically, e.g. hCAT-1-mediated influx and efflux are markedly reduced at low concentrations of trans-substrate, whereas influx and efflux mediated by hCAT-2A are unchanged (10). In the present study, we performed efflux experiments to determine the sensitivity of the chimeric proteins to trans-stimulation because the concentration of cationic amino acids can be defined more precisely in the extracellular buffer compared with the cytosol. The concentration of 1 mM trans-L-arginine was chosen for these experiments because our previous results demonstrated that trans-stimulation of hCAT-1 is saturated at 100 µM L-arginine and that hCAT-2A is largely independent of extracellular L-arginine up to 10 mM (6, 8). Our results with the hCAT-2A/1.BK and hCAT-2A/1.NK chimeras demonstrate that the 69-amino acid segment encoded by the NcoI/KpnI fragment determines the sensitivity of hCAT-2A to trans-stimulation, whereas the short part encoded by the BamHI/NcoI fragment does not seem to contribute. However, we were not able to pinpoint the amino acid residues within the 69-amino acid fragment that are responsible for the sensitivity to trans-stimulation. The results with the hCAT-2A/1.SK chimera (carrying TM X of hCAT-1 in the hCAT-2A backbone) suggest that TM X has an influence on trans-stimulation. However, hCAT-2A/1.SK and hCAT-2A(R369E) both exhibited an increased sensitivity to trans-stimulation compared with hCAT-2A, but the sensitivity of hCAT-2A/1.SK(R369E) was not further increased. Also, the sensitivity of the double mutant hCAT-2A(R369E/N381i) to trans-stimulation was not different from that of hCAT-2A, suggesting that the insertion of the asparagine residue desensitizes the protein to trans-stimulation. A shortcoming of these experiments lies in the low efflux activities of hCAT-2A/1.SK and all hCAT-2A mutants, making the interpretation of these results difficult, as a small reduction in the absolute efflux rate at no trans-substrate is sufficient to suggest an increased sensitivity to trans-stimulation. The hCAT-2A(R369E/N381i) mutant, which exhibited the same substrate affinity as hCAT-1, but no sensitivity to trans-stimulation, demonstrates that substrate affinity and sensitivity to trans-stimulation can be separated.
In summary, our study provides important insights into the structure/function relation of the hCAT proteins. We have identified two amino acid residues that determine the apparent substrate affinity of CAT-2A on both sides of the membrane. These residues are not localized within a transmembrane domain and therefore are not likely to be part of the substrate-binding site and translocation pathway. Their localization in the fourth intracellular loop suggests that this protein domain might control the affinity of the substrate-binding sites indirectly, e.g. by influencing their conformation. The two amino acid residues are within the 42-amino acid fragment where the sequence of hCAT-2A diverges from the sequence of hCAT-2B and that extends into TM IX. This fragment also influences the sensitivity of hCAT-2A to trans-stimulation. However, other amino acid residues and a more complex interaction between different amino acid residues within this area must be involved. In addition, the adjacent TM X seems to also play a role in determining the sensitivity to trans-stimulation. These findings, together with the observation that subtle changes in the sequence of TM X lead to loss of function in hCAT-1, led us to assume that TMs IX and X might well be part of the translocation pathway.
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FOOTNOTES
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* This work was supported by Grant Cl 100/3-4 and Collaborative Research Center Grant SFB 553 (Project B4) from the Deutsche Forschungsgemeinschaft (Bonn, Germany). 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. 
To whom correspondence should be addressed. Tel.: 49-6131-39-33178; Fax: 49-6131-39-36611; E-mail: Closs{at}mail.uni-mainz.de.
1 The abbreviations used are: CAT, cationic amino acid transporter (prefixes m and h represent mouse and human, respectively); TM, transmembrane domain; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline. 
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