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PHYSIOLOGICAL REVIEWS   Vol. 78 No. 4 October 1998, pp. 969-1054
Copyright ©1998 by the American Physiological Society

Molecular Biology of Mammalian Plasma Membrane Amino Acid Transporters

MANUEL PALACÍN, RAÚL ESTÉVEZ, JOAN BERTRAN, AND ANTONIO ZORZANO

Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, and Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncològica, Barcelona, Spain

I. INTRODUCTION
    A. Amino Acid Transport Systems in the Plasma Membrane of Mammalian Cells
    B. Strategies Used to Identify Mammalian Amino Acid Transporters as Yet Uncloned
II. CURRENT KNOWLEDGE OF THE MOLECULAR STRUCTURE OF AMINO ACID TRANSPORT SYSTEMS
    A. Cationic Amino Acid Transporters
    B. Superfamily of Sodium- and Chloride-Dependent Neurotransmitter Transporters
    C. Superfamily of Sodium-Dependent Transporters for Anionic and Zwitterionic Amino Acids
    D. Putative Subunits of Sodium-Independent Cationic and Zwitterionic Amino Acid Transporters
III. INHERITED DISEASES OF PLASMA MEMBRANE AMINO ACID TRANSPORT
IV. PROSPECTS
REFERENCES

    ABSTRACT
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Palacín, Manuel, Raúl Estévez, Joan Bertran, and Antonio Zorzano. Molecular Biology of Mammalian Plasma Membrane Amino Acid Transporters. Physiol. Rev. 78: 969-1054, 1998. --- Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x-c) and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

    I. INTRODUCTION
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Amino acid transport across the plasma membrane mediates and regulates the flow of these ionic nutrients into cells and, therefore, participates in interorgan amino acid nutrition. In addition, for specific amino acids that act as neurotransmitters, synaptic modulators, or neurotransmitter precursors, transport across the plasma membrane ensures reuptake from the synaptic cleft, maintenance of a tonic level of their extracellular concentration, and supply of precursors in the central nervous system (for review, see Refs. 93, 96, 97, 505). Transfer of amino acids across the hydrophobic domain of the plasma membrane is mediated by proteins that recognize, bind, and transport these amino acids from the extracellular medium into the cell, or vice versa. In the early 1960s, after the pioneer work of Halvor N. Christensen's group, different substrate specificity transport systems for amino acids in mammalian cells (mainly erythrocytes, hepatocytes, and fibroblasts) were identified (reviewed in Ref. 96), and general properties of mammalian amino acid transport were revealed: stereospecificity (transport is faster for L-stereoisomers) and broad substrate specificity (several amino acids share a transport system). Since these initial studies, the main functional criteria used to define amino acid transporters have been 1) the type of amino acid (acidic, zwitterionic, or basic as well as other characteristics of the side chain) the protein moves across the membrane, according to substrate specificity and kinetic properties, and 2) the thermodynamic properties of the transport (whether the transporter is equilibrative or drives the organic substrate uphill). This functional classification has been retained to date, since structural information on higher eukaryote amino acid transporters is incomplete.

Isolation of the first brain GABA transporter (184) in 1990 and the identification of the first cationic amino acid transporter in 1991 (281, 590) represent the starting points for the cloning of mammalian amino acid transporter genes. Several strategies have been used to identify mammalian amino acid transporters. During the 1980s, several groups attempted the purification of these transporters by different methods. Purification strategies have yielded few structural data, although for a couple of transporters related to neurotransmission (the GABA transporter GAT-1 and the glutamate transporter GLT-1), these data allowed microsequencing or generation of specific antibodies that have been used to isolate cDNA clones (184, 424). Alternative strategies and serendipity allowed the identification of up to 22 cDNA (including splice variants, but not species counterparts) for mammalian amino acid transporters or related proteins (see sect. II). This structural information is not complete. The genes identified seem to correspond to eight classic transport systems and their variants, whereas another eight of the major amino acid transport systems are unknown at the molecular level (see Table 1). An excellent review by Kilberg's group (342) describes the molecular biology of the amino acid transporters cloned up to 1995. 

 
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  		TABLE 1.   Best-known amino acid transport systems present in the plasma membrane of mammalian cells

The molecular identification of amino acid transporters or related proteins leads to ongoing studies on the structure-function relationship and the molecular genetics of the pathology associated with these transporters. In this review, attention is paid to the molecular biology, structure-function relationship, physiological role, and human genetics of amino acid transporters. Regulation of plasma membrane amino acid transport in mammals is beyond the scope of the present review and has been extensively reviewed (96, 278, 350).

A. Amino Acid Transport Systems in the Plasma Membrane of Mammalian Cells

Functional studies based on saturability of transport, substrate specificity, kinetic behavior, mode of energization, and mechanisms of regulation performed in perfused organs, isolated cells, and purified plasma membranes led to the identification of a mutiplicity of transport agencies in the plasma membrane of mammalian cells (for review, see Refs. 26, 94-96). The properties of some of the best-characterized amino acid transport systems are summarized in Table 1. From these studies it is evident that a particular transport system carries different amino acids and that amino acid transport systems show overlapping specificities. Different cells contain a distinct set of transport systems in their plasma membranes, as a combination of common or almost ubiquitous (e.g., systems A, ASC, L, y+ and X -AG ) and tissue-specific transport systems (e.g., systems Bo,+, Nm, and bo,+ as well as variants of common transport systems). It has been proposed that this arrangement permits both fine regulation of substrate and cell-specific amino acid flows and economy in the number of structures mediating cellular and interorgan amino acid fluxes (96, 97).

1. Common systems for zwitterionic amino acids

The zwitterionic amino acid transport systems A, ASC, and L are present in almost all cell types. Systems A and ASC mediate symport of amino acid with small side chain with sodium, and system L mediates uniport of amino acids with bulky side chains (i.e., branched and aromatic groups). System L has a high exchange property (i.e., it shows trans-stimulation, stimulation by substrates in the trans-compartment) and has been postulated to serve in many circumstances to mediate efflux from cells rather than influx into cells (96). Preferred substrates for system A are alanine, serine, and glutamine, and for system ASC alanine, serine, and cysteine (99, 101). In contrast, amino acids with a small, nonbranched side chain are poor substrates for system L, for which the analogs 2 - a m i n o e n d o b i c y c l o - [ 2, 2, 1 ] - h e p t a n e - 2 - c a r b o x y l i c   a c i d (BCH) and 3-aminoendobicyclo-[3,2,1]-octane-3-carboxylic acid (BCO) are model substrates in the absence of sodium. System L has been subdivided into subtypes L1 (substrate affinity in the micromolar range) and L2 (substrate affinity in the millimolar range), which are present at different ages in hepatocytes and hepatoma cell lines (597). Therefore, zwitterionic amino acids with bulky side chains are not cotransported with sodium in many cell types. This is different in epithelial cells, where broad specificity systems (e.g., systems Bo, Bo,+) accumulate these amino acids by coupling the electrochemical gradient of sodium. One criterion for discrimination of system A is the fact that it transports N-methylated amino acids (101); in many instances, sodium-dependent transport of alanine inhibitable by the model analog N-methylaminoisobutyric acid (MeAIB) or the sodium-dependent transport of MeAIB is used for determining system A transport activity. System A is highly pH sensitive. Intracellular histidyl residues may form part of the pH sensor of the transporter (43, 384). In contrast, system ASC is relatively pH insensitive. Additional differential characteristics of systems A and ASC are that the former is electrogenic and shows the property of trans-inhibition (i.e., inhibition by substrates in the trans-compartment), and the latter may be electroneutral and shows trans-stimulation, probably as a consequence of a high property of exchange. Therefore, whereas system A is considered a true sodium-amino acid cotransporter, system ASC may be an antiporter of amino acids necessarily associated with the movement of sodium in both directions (for review, see Ref. 185). Transporters ASCT1-2 from the superfamily of sodium- and potassium-dependent transporters of anionic and zwitterionic amino acids may represent transporter variants of system ASC (see sect. II). The molecular identity of systems A and L is unknown.

An important distinguishing feature of system A is that in many cell types its activity is highly regulated. This includes upregulation during cell-cycle progression and cell growth in many cell types, and hormonal control (insulin, glucagon, catecholamines, glucocorticoids, and growth factors and mitogens) through a wide variety of mechanisms. Thus glucagon and epidermal growth factor short-term stimulate system A in hepatocytes, whereas insulin upregulates system A in a gene transcription-dependent manner in hepatocytes and through a rapid mechanism in skeletal muscle. This is independent of protein synthesis and the electrochemical gradient of sodium, suggesting direct stimulation of preexisting system A transporters or recruitment of these to the plasma membrane. Paradoxically, situations associated with insulin deficiency or insulin resistance (e.g., diabetes, starvation, pregnancy) are associated with upregulation of system A activity in both hepatocytes and skeletal muscle. Two additional long-term maximum velocity (Vmax) regulation processes of system A have been demonstrated only in culture or incubated cells: adaptative regulation (i.e., repression and derepression of system A in the presence or absence of amino acids) and upregulation by hyperosmolarity. In both types of regulation, there is indirect evidence for the presence of system A regulatory proteins. For a detailed description of the putative mechanisms involved in the regulation of system A, the reader is directed to excellent recent reviews (185, 201, 350, 414) and to specific articles on insulin action in skeletal muscle (188-190, 561). The complexity of the regulation of system A emphasizes the importance of this transport activity in maintaining the accumulation of small side-chain zwitterionic amino acids as a source for precursor molecules, energy, and perhaps even as osmolytes. Unfortunately, the lack of structural information on the system A transporter (see sect. IB1) prevents further understanding of the molecular mechanisms underlying system A regulation.

2. Tissue-specific systems for zwitterionic amino acids

Additional sodium-dependent zwitterionic transport systems of restricted distribution have been described (Table 1). Transport of L-glutamine, L-histidine, and L-asparagine in hepatocytes has been demonstrated to occur via a sodium-dependent transport system named N (276). A system with similar properties to system N was defined in skeletal muscle and termed Nm (222). System Gly, specific for glycine and sarcosine, occurs in several cell types (134). Transporters GLYT1-2, from the superfamily of sodium- and chloride-dependent transporters of neurotransmitters, may represent variants of system Gly (see sect. II). A transport system for beta -alanine, taurine, and GABA (system BETA) has been characterized in several tissues and with differences in substrate affinity and specificity (369). Transporters GAT1-3, BGT-1, and TAUT, which belong to the superfamily of sodium- and chloride-dependent transporters of neurotransmitters, may be considered variants of system BETA (see sect. II).

Several zwitterionic transport systems seem to be specific to the apical pole of epithelial cells. In intestinal brush-border membrane vesicles, a sodium-dependent transport system (NBB, which stands for neutral brush border) serving for neutral amino acids is present. This system was renamed B for consistency with other broad-specificity systems (e.g., Bo,+, bo,+, b+; see below) (337). More recently, in bovine renal brush-border membranes, a sodium-dependent system for neutral amino acids was described that was termed Bo (329). Most probably the transport systems B (NBB) and Bo represent the same transport agency distributed in epithelial cells, as suggested after the cloning of the putative transporter for system Bo (ATBo, from the superfamily of sodium- and potassium-dependent transporters for anionic and zwitterionic amino acids; see sect. II). This broad-specificity system is thought to be responsible (together with the dipeptide and tripeptide transport systems; for review, see Refs. 122, 299) for the bulk of renal reabsorption and intestinal absorption of zwitterionic amino acids. Therefore, system Bo and the ATBo cDNA could represent the transport activity and the corresponding transcript that are altered in Hartnup disease (304), an inherited hyperaminoaciduria of neutral amino acids (see sect. III). Additional transport systems (see Table 1) seem to be specific to brush-border membranes. System IMINO, which catalyzes sodium-dependent transport of proline and N-methylated glycines and is inhibited by MeAIB, has been detected in kidney and intestine and in other epithelia (192, 376, 514). The proline transporter PROT, from the superfamily of sodium- and chloride-dependent transporters of neurotransmitters, might represent a brain-specific high-affinity [Michaelis constant (Km) in the low micromolar range] variant of system IMINO; this is not yet clear (see sect. II). The broad-specificity (cationic and zwitterionic amino acids) transport systems Bo,+ and bo,+ are described in section IA3, together with cationic amino acid transport systems.

3. Cationic amino acid transport systems

Five transport systems that mediate the uptake of cationic amino acids are known (Table 1). One corresponds to the widespread classical system y+, whereas the other four were discovered in the late 1980s and early 1990s (systems y+L, b+, bo,+, and Bo,+) and at present have been described only in specific tissues. The activity of all these systems can be distinguished by their affinity for the cationic amino acids, by their dependence on sodium, and by their capacity to share transport with zwitterionic amino acids (for a short review, see Ref. 105). System y+ catalyzes high-affinity (Km in the micromolar range) sodium-independent transport of cationic amino acids and the transport of zwitterionic amino acids with low affinity (Km in the large millimolar range; affinity increases with chain length) only in the presence of sodium; this system is electrogenic and accumulates its substrates by coupling with the cell plasma membrane potential (98, 455, 601). System y+L was discovered in human erythrocytes (127) and has also been described in placenta (135, 146). It is possible that system y+L is widely distributed among different tissues; thus it has also been detected in human fibroblasts (D. Torrents and M. Palacín, unpublished data). This system transports cationic amino acids with high affinity (Km in the micromolar range) with no need for sodium in the external medium, but it transports both small and large zwitterionic amino acids with high affinity (Km in the micromolar range) in the presence of external sodium; in the absence of sodium, transport of zwitterionic amino acids is of very low affinity. In addition to this, system y+ and y+L activities could be discriminated, at least in erythrocytes and placenta, by N-ethylmaleimide (NEM) treatment, the former being sensitive and the latter resistant. Systems y+ and y+L show very high capacity for trans-stimulation (i.e., exchange). It is thought that exchange via system y+ allows equilibration of cationic amino acids across the plasma membrane, whereas heteroexchange between cationic and zwitterionic amino acids plus sodium via system y+L catalyzes the efflux of cationic amino acids against the membrane potential, the driving force being provided by the sodium ion concentration gradient (14, 90). Systems y+ and y+L have been suggested to be candidate transport activities affected in the inherited disease lysinuric protein intolerance (LPI) (for review, see Ref. 497). This is discussed in section III. The cDNA for up to five potential y+ transporters have been isolated (CAT-1, CAT-2, the splice variant CAT-2a, CAT-3, and possibly the recently identified CAT-4), which form the cationic amino acid transporter family (see sect. II). Several groups described expression of system y+L transport activity in oocytes by the 4F2hc surface antigen, which shows homology with another protein, rBAT, also related to broad-specificity amino acid transport. Because 4F2hc and rBAT are less hydrophobic than typical transporter proteins and form disulfide-bound heterodimers with unidentified proteins, it has been suggested that 4F2hc may represent a putative subunit of system y+L transporter; this ascription is not yet clear (see sect. II).

Systems Bo,+, bo,+, and b+ were discovered in mouse blastocysts (576-578; for review, see Ref. 573). Among the systems that form the series of transport activities for cationic amino acids, the embryonic sodium-independent systems b+ (subtypes b1+ and b2+, which differ in the embryonic stage expression and sensitivity to cationic amino acid inhibition) show the narrowest specificity, serving only for cationic amino acids (576). Systems Bo,+ and bo,+ show very similar broad specificity with high affinity (Km in the micromolar range) for cationic and small and large zwitterionic amino acids. As a distinguishing feature, the former is sodium dependent and inhibitable by BCH and BCO, and the latter prefers bulky alpha ,beta -unbranched zwitterionic amino acids. More probably, both systems have a wide distribution on epithelial cells. In fact, system bo,+ (or a variant, bo,+-like) has been detected in renal epithelial cells and in Caco-2 cells (374, 557). Expression of rBAT, a protein homologous to the cell surface antigen 4F2hc, is needed for system bo,+-like transport activity; it is believed that rBAT acts as a subunit of this transporter (see sect. II). Mutations in rBAT/system bo,+-like transport activity cause cystinuria type I (for reviews, see Refs. 170, 408, 409), an inherited hyperaminoaciduria due to defective renal reabsorption and intestinal absorption of cationic amino acids and cystine (for review, see Ref. 487). The role of rBAT in cystinuria is discussed in section III.

4. Anionic amino acid transport systems

L-Glutamate and L-aspartate are accumulated in many cells (e.g., neurons and glial cells, hepatocytes, enterocytes, fibroblasts, and placental trophoblasts) by the high-affinity (Km in the micromolar range) sodium- and potassium-dependent system X -AG (165) (for review, see Ref. 185). This system shows identical affinity for the D- and L-stereoisomers of aspartate (165). Variants of this transport systems occur in neural tissues (100, 147). It is believed that the five glutamate transporters EAAT1-5 from the superfamily of sodium- and potassium-dependent transporters of anionic and zwitterionic amino acids represent variants of system X -AG (see sect. II).

Several cell types (e.g., hepatocytes, fibroblasts, and embryonic cells) transport L-glutamate (specifically anionic amino acids with 3 or more carbon atoms in the side chain) and L-cystine (as tripolar ion) via the sodium-independent antiport system x -C (28, 533). This system has an apparent Km in the 100-200 µM range, is insensitive to membrane potential, and presents trans-stimulation (for review, see Ref. 185). Bannai's group (27) proposed that this system participates in a glutamine-cystine cycle that helps cells to resist oxidative stress; glutamine, entering the cell via systems ASC and A, is converted to glutamate, which is exchanged for cystine via the oxidative stress-induced system x -C; accumulated cystine then nourishes glutathione synthesis, which protects cells against oxidative insult (27, 29, 596; for review, see Ref. 228).

B. Strategies Used to Identify Mammalian Amino Acid Transporters as Yet Uncloned

As described in section IA, the present explosion of cloned cDNA related to plasma amino acid transport in mammals is revealing an intriguing range of structural diversity within amino acid transporter proteins. This diversity is already even more pronounced than that shown by the sodium-dependent glucose transport (for review, see Ref. 608) and the facilitated glucose transporter isoforms (for review, see Refs. 381, 556). The lack of high-affinity inhibitors for mammalian amino acid carriers and their low abundance in plasma membranes complicate their structural identification and isolation. Because of this, there are many amino acid transport systems not yet identified at a molecular level (see Table 1). Among these systems, there are the highly regulated system A and the well-characterized systems L, N, and x -C. In this section, we focus on the strategies used to identify these four transporters and also speculate, in some cases, about why some strategies have failed.

1. System A

One of the goals of several laboratories has been to reconstitute and purify system A transporter. Kilberg's group (195) reported the solubilization, reconstitution, and partial purification of system A (70-fold over plasma membrane vesicles). They then used this protein fraction to immunize mice for the generation of monoclonal antibodies. Some of these antibodies specifically coprecipitate fodrin and system A transport activity. Because the protein ankyrin often binds directly to integral membrane proteins and fodrin, the authors tested whether an antibody against ankyrin could immunoprecipitate system A transport activity, and it did. McGivan's group (437) partially purified system A activity from rat liver with concanavalin A-affinity chromatography. This demonstrated that either system A or a protein bound to the carrier is a glycoprotein. McCormick and Johnstone (348) purified system A transport activity from Ehrlich ascites with a 30-fold enrichment. Three major peaks were eluted from the system A-purified Ehrlich cell preparation: high-molecular-mass aggregates, a low-molecular-mass band (~40 kDa), and the most conspicuous band of 120-130 kDa. Interestingly, polyclonal antibodies against the 120- to 130-kDa purified fraction immunoprecipitated system A transport activity. More recently, NH2-terminal sequence analysis of the 120- to 130-kDa peptide revealed a sequence similar to that of the alpha 3-subunit of the alpha 3beta 1-integrin (349). Further purification of these extracts using lectin columns resulted in the separation of most of the alpha 3beta 1-integrin from the system A activity, indicating that this integrin is not essential for amino acid transport. Moreover, the fact that transfection of alpha 3-integrin into K562 or RD cells increased system A transport activity provides evidence that this protein could modulate this transporter. In summary, from these studies of reconstitution, one may conclude that several distinct proteins contribute to the entire system A transport activity. Probably in the near future the microsequencing of some of the proteins present in these purified extracts will result in the isolation of all the components necessary for system A function.

Another strategy used to identify system A is the functional expression in Xenopus laevis oocytes. Expression of system A has been claimed (409, 546) based on the following (for review, see Ref. 277). 1) The effect of glucagon on system A in vivo was maintained after mRNA extraction and injection into the oocyte; glucagon is known to stimulate system A transport activity in liver, at least in part, through mRNA and protein synthesis-dependent mechanisms. 2) The apparent substrate affinities reported by these authors were in the same range as those described for the transport of the substrates tested via system A. 3) The cis-inhibition of the transport of L-alanine induced by MeAIB suggested that at least part of this expressed activity was due to system A. 4) The expressed transport activity, in contrast to the endogenous uptake, was inhibited by an extracellular pH of 6.5. 5) Messenger RNA from the Chinese hamster ovary (CHO) cell line alar-H3.9, which overexpresses system A activity (371, 372), resulted in higher transport rates than mRNA from the parental cell line CHO-K1. More recently, Lin et al. (313) presented evidence that mRNA of differing sizes (2.2 and 4.2 kb) from two cell lines (GF-14 cells, Ehrlich cells) increase the expression of system A transport upon injection into Xenopus oocytes. However, only the synthesis of the 2.2-kb transcript is raised by insulin, which is consistent with the idea that there are variants of system A transporter (see below). Expression cloning of this transport activity has not yet been achieved, possibly due to the high background (i.e., basal oocyte endogenous activity), and perhaps because the system A transporter would need the expression of different proteins that form part of the whole transporter or are upregulators of its activity (e.g., as shown by the adaptative or osmotic regulation of system A; see above, for review see Ref. 350). In the same line of functional expression of system A transport activity, Lin et al. (312) restored normal growth of a mutated yeast cell line incapable of growth in minimal medium with proline by transfection with a cDNA (E51) from mouse Ehrlich cells. This cDNA is 90% homologous to gamma -actin. Similarly, this cDNA increases sodium-dependent amino acid uptake when expressed in oocytes and in a mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport activity. This suggests that the gamma -actin-like protein coded for by E51 cDNA may play a significant regulatory role in sodium-dependent amino acid transport. In summary, reconstitution-purification and functional expression studies suggest that a multiplicity of proteins might be involved in the functional expression and modulation of system A transport activity.

Another approach is the development of cell lines that show mutations in amino acid transport activity (reviewed in Refs. 113, 603). In this way, Englesberg's group (371) has isolated constitutive or derepressed mutants for system A activity from CHO-K1 cells by alanine-resistant selection for proline uptake (alar4 mutant) and by a stepwise selection (hydroxyurea treatment and resistance to increased alanine concentration) (alar4-H3.9 mutant). In comparison to the wild type, these mutants showed higher system A activity in isolated plasma membrane vesicles and higher mRNA-induced sodium-dependent aminoisobutyric acid uptake in Xenopus oocytes (371, 546). These mutants have increased levels of peptides banding at 62-66 kDa and 29 kDa. Sequencing the NH2 terminus of the 62- to 66-kDa peptide shows between 80 and 100% identity with the mammalian mitochondrial 60-kDa heat-shock protein (HSP60) (235). Whether these proteins are components of system A carrier is at present unknown.

Other approaches involve the chemical modification of specific residues by covalent reagents. Hayes and McGivan (202) identified a 20-kDa protein as a putative component of sodium-dependent alanine transport in liver plasma membrane vesicles. On the other hand, the presence of histidine residues critical for activity in the system A from rat liver (i.e., sensitive to diethyl pyrocarbonate inactivation) has also been demonstrated using this approach (43). Thiol reagents have been used to reveal structural differences between these carriers and between normal and transformed cells. It has been suggested that structural differences occur in system A transporters of transformed cells, based on the much greater sensitivity to NEM inactivation of liposome-reconstituted system A activity from normal hepatocytes than that from hepatoma cell lines (132). Moreover, all these studies should be critically evaluated, since these specific reagents can modify, with different affinities, a variety of different amino acid residues in proteins. In any case, no further structural information on system A has been achieved with this strategy.

2. System L

Using the strategy of functional expression in Xenopus oocytes, Oxender's group (521) reported the expression of a sodium-independent L-leucine transport system shared with dibasic amino acids, by injection of mRNA from CHO cells. This suggests that the expressed transport could correspond to that induced by the expression of rBAT (i.e., system bo,+) and not to system L. In any case, this ascription is not yet clear, since the data from Oxender's group (521) and rBAT-expressed amino acid transport activity (549) differ in the sensitivity to inhibition by L-tryptophan. More recently, Broër et al. (68) also expressed sodium-independent isoleucine transport activity from mRNA of rat brain in oocytes. The sodium-independent component of isoleucine transport was inhibited by leucine, phenylalanine, and BCH, consistent with the expression of a system L-like transporter. However, the isolated cDNA responsible for this activity was rat 4F2hc, which also expresses cationic amino acid transport in oocytes (69). As discussed in section II, several groups proposed that 4F2hc expresses a system y+L-like transport activity in oocytes. In our view, expression of system L in oocytes has not been conclusively demonstrated.

Another interesting strategy to assess the structure of this transporter has been developed by Oxender's group (136). It is based on the fact that the transport activity of system L can be derepressed by severe "starvation" for leucine or by increasing the temperature of culture in mutant cell lines with temperature-sensitive leucyl-tRNA synthetase (reviewed in Ref. 603). Oxender's group (136) transformed a temperature-sensitive leucyl-tRNA synthetase mutant CHO cell line (CHO-025C1) with human DNA from a cosmid library. Subsequent selection of transformants for inability to grow above the permissive temperature in the presence of low leucine concentration allowed the isolation of cells with higher (<2-fold) leucine uptake activity. To date, no report has described the rescue of the human DNA sequences responsible for the above-mentioned transformation.

More recently, Segel's group (606) has developed a new strategy to identify the carrier protein(s) responsible for mammalian L-system amino acid transport. In chronic lymphocytic leukemia (CLL), B lymphocytes have markedly disminished L-system transport, which is restored after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). These authors identified six candidate L-system-related proteins in TPA-treated CLL cells using an L-system photoprobe (iodoazidophenylalanine) and ultra-high-resolution two-dimensional gel electrophoresis. Two of these six proteins were microsequenced and show sequence similarity to the mitochondrial heat-shock protein (HSP60). This report and the data published by Englesberg's group (235) implicate the family of heat shock proteins in the regulation of some transport processes. How these proteins develop their function in systems A and L transport activity is unknown.

3. System N

Kilberg's group (143), with the same approach of aggregation and differential solubility used to purify system A, achieved a 600-fold enrichment for system N amino acid transport activity in reconstituted proteoliposomes (540). They identifed a 100-kDa protein involved in system N amino acid transport activity by generating monoclonal antibodies against the purified fraction that immunoprecipitate system N transport activity (539). These tools may allow the identification of system N transporter.

Rennie's group (551) has reported the expression of rat liver glutamine transporters after injection of rat liver mRNA into Xenopus oocytes. They attributed part of the L-glutamine-induced transport activity to system N based on a characteristic feature of this transport system, that is, the toleration of lithium by sodium substitution and the inhibition by L-histidine in lithium medium. By size-fractioning the mRNA, they found three different induced transport activities: one sodium independent induced by 2.8-3.6 kb mRNA, another sodium-dependent, lithium sustitution intolerant induced by 1.9-2.8 kb mRNA, and one induced by a lighter fraction (<1.9 kb) that is sodium or lithium dependent and that could correspond to system N.

4. System x -c

Bannai's group (227), working with manually defolliculated oocytes, has reported the expression of a mouse-macrophage cystine transporter with characteristics of system x -c, as it was sodium independent and glutamate inhibitable. Fractions of mRNA of 1.5-2.9 kb are responsible for this induction. Although oocytes seem to express an endogenous system x -c (574), expression of this system correlates with injection of mRNA from x -c-rich cells (macrophages stimulated by diethylmaleate in culture), but not from x -c-poor cells (noncultured macrophages and mouse leukemia L1210 cells). In addition, cystine uptake expressed by diethylmaleate-stimulated macrophage mRNA was, in contrast to the endogenous cystine uptake, pH sensitive, highly temperature sensitive, and inhibitable by glutamate. This line of research may lead to the identification of system x -c transporter.

    II. CURRENT KNOWLEDGE OF THE MOLECULAR STRUCTURE OF AMINO ACID TRANSPORT SYSTEMS
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In this section, the cDNA clones that have been related to plasma membrane amino acid transport in mammals are discussed from the structural and functional points of view. With regard to the primary structures elucidated to date, it can be summarized that two main types of membrane proteins are involved in amino acid transport: those that present multiple (i.e., 10-14) transmembrane domains and are therefore considered as putative transporters and those that do not fit this general model and are considered as activators or as components of oligomeric transporters. The first group with typical structure of transporters is arranged in families of related cDNA: 1) the family of sodium-independent cationic amino acid transporters (CAT); 2) superfamily of sodium- and chloride-dependent neurotransmitter transporters, which includes amino acid transporters; and 3) the superfamily of sodium-dependent, and in some cases potassium-dependent, anionic or zwitterionic amino acid transporters. The second group corresponds to the family of proteins rBAT and 4F2hc that induce sodium- and chloride-independent amino acid transport with broad specificity (i.e., for dibasic and zwitterionic amino acids) in X. laevis oocytes. The members of this family are less hydrophobic than typical transporters and show heteroligomeric structure.

Another protein related to amino acid transport, whose primary structure was elucidated many years ago, is the anion exchanger band 3 (290). Although its function as an anion exchanger is well accepted, it has been shown to be associated with the transport of some amino acids (e.g., glycine, taurine, and beta -alanine), especially under conditions of hyposmotic stress; in response to swelling, erythrocytes recover their initial volume by releasing organic osmolytes via a pathway with a pharmacology similar to that of band 3 (114, 203, 308). This amino acid transport has the properties of a volume-sensitive size-limited anion channel (171). Interestingly, expression of trout band 3 in oocytes resulted in anion-exchange activity but also in chloride channel activity and taurine transport (150). At present, it is not known whether band 3 is involved in the amino acid channel formation or in its regulation. The role of band 3 in amino acid transport is outside the scope of this review. This and the molecular biology of band 3 have recently been reviewed (10, 380, 574).

A. Cationic Amino Acid Transporters

Four homologous human and rodent genes defining a family of cationic amino acid transporters (CAT-1, -2, -3, and -4) have been, or are in the process of being, identified (Table 2). First, expression in oocytes revealed the ecotropic murine leukemia virus receptor (9) (now named CAT-1) as a putative cationic amino acid transporter (281, 590). Second, full-length cDNA cloned from a previously identified murine T-lymphoma cell line cDNA (Tea, for "T early activation" gene; Ref. 334) showed significant homology with CAT-1 and cationic amino acid transport expression in oocytes (106, 108, 242, 448). These cDNA, now named CAT-2 and CAT-2a, represent splice variants (the "high-affinity" or "T-cell" variant CAT-2 and the "low-affinity" or "liver" variant CAT-2a) (see Table 2) of a single gene (336). It has been suggested that the three putative proteins (CAT-1, CAT-2, and CAT-2a variants) may contain 14 transmembrane domains (TM) (9, 106) (Figs. 1 and 2, see below). Very recently, with strategies based on sequence homology with CAT-1, the mouse and rat counterparts (95% amino acid sequence identity between them) of a new brain-specific cationic amino acid transporter, CAT-3, have been isolated (219, 229). Very recently, Sebastio and co-workers (484, 485) identified a human EST sequence (Table 2) with significant homology to the 5'-end of the open reading frame of CAT-1 and CAT-2/2a cDNA (Fig. 1). Screening of the full-length human CAT-4 cDNA [we renamed HCAT-3 (485) as CAT-4 after the reported cloning of rat and mouse CAT-3 (219, 229)] has been completed, and the putative protein shows 34-38% identity with human CAT-1, -2, and -3 (G. Sebastio, personal communication). At present, there are no reported data on the transport activity associated with CAT-4 expression. Screening, performed by us (DBEST, December 1996), for additional expressed sequence tag sequences homologous to CAT transporters indicative of additional members of the family was negative.

 
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  		TABLE 2.   Human cationic amino acid transporters


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  		FIG. 1.   Amino acid sequence comparison of human cationic amino acid transporters (CAT). HO5853 sequence corresponds to best EST sequence in database DBEST that showed homology with human CAT-1 and CAT-2, and rat CAT-3, and that served for human CAT-4 cloning (G. Sebastio; personal communication). Amino acid residues present in all CAT transporter sequences known to date are indicated by gray boxes. Straight lines over sequences indicate putative transmembrane (TM) domains (I-XIV). Between TM V and TM VI, two potential N-glycosylation sites are conserved (open boxes). Dash lines indicate gaps for sequence alignment, which corresponds to that reported by Closs (105).


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  		FIG. 2.   Fourteen TM transmembrane topology model for murine mCAT-2 transporter. Extracellular (EL) and intracellular (IL) loops are numbered. Amino acid residues conserved in all known CAT transporters are indicated in gray circles, and residues that are specific for known CAT-2 and CAT-2a transporters are indicated in white over a black circle. At bottom, 41- to 42-amino acid segment corresponding to splice variant exon of mCAT-2 and mCAT-2a transporters are compared with homologous mCAT-1 and mCAT-3 regions (residues present in at least 3 of sequences are contained in gray boxes, and those specific for low-affinity CAT-2a isoform are in white on black boxes). In EL3 loop, amino acid sequence that binds viral envelope glycoprotein gp70, present in murine and rat CAT-1, is boxed. Three residues, a Glu in TM III and 2 glycosylated (Y) Asn residues in EL3, are marked by squares, indicating homologous residues that have been analyzed by site-directed mutagenesis studies of mCAT-1 sequence. Human CAT-1 gene structure has been published (619), and it has been reported that mouse CAT-1 gene contains 8 coding exons (419). To our knowledge, gene structure of CAT-2 has not been reported.

Figure 1 compares the amino acid sequences of the putative human CAT-1 and CAT-2a proteins, the mouse CAT-3 protein, and the putative NH2-terminal fragment of the human CAT-4 transporter. Two potential N-glycosylation sites, conserved in all known sequences, are located in the extracellular loop EL3 in the 14-TM model (Figs. 1 and 2). The known amino acid sequences of CAT-2 and CAT-2a (98% identity between the murine counterparts; see Fig. 2) are ~60% identical to that of CAT-1 (106, 448). The rat and mouse CAT-3 show 53-58% amino acid sequence identity with the isolated CAT-1, CAT-2, and CAT-2a cDNA (219). Two regions of extensive amino acid sequence identity (7E80%) of ~150 and 200 amino acid residues, comprising the first three transmembrane domains and transmembrane domains VI-X, are present in the CAT-1, -2, and -3 proteins (334; see Figs. 1 and 2). Murine CAT-2 and CAT-2a differ only in a 41- to 42-amino acid segment located in this highly conserved region (intracellular loop IL4 between TM domains VIII and IX of the 14-TM model) (Fig. 2). Because a single genomic fragment contains both exons, the isoforms result from mutually exclusive alternate splicing of the primary trancript (unpublished data from MacLeod and co-workers quoted in Ref. 336). This amino acid sequence region has a role in substrate binding, as demonstrated by the expression of CAT-2/CAT-2a chimeric transporters (backbone and the 42-amino acid domain) (108).

The CAT transporters are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC family) that catalyze solute uniport, solute/cation symport, or solute/solute antiport in yeast, fungi, and eubacteria (448). Marked sequence divergence of these proteins was observed mainly in the hydrophilic NH2 terminals that precede the first transmembrane helices and in the COOH-terminal regions (448). Southern blot studies revealed that all vertebrates examined hybridize to the probes of CAT-1 and CAT-2, indicating a high conservation of these proteins among vertebrates (448). Thus the human CAT-1 protein (7, 619) and the rat CAT-1 protein (433, 610) are 86 and 95% identical, respectively, to the mouse CAT-1, and the human analogs of CAT-2 and CAT-2a proteins are ~90% identical to the murine counterparts (218; unpublished data quoted in Ref. 105).

The chromosomal location of the human CAT genes is showed in Table 2. The possible involvement of CAT genes in LPI, a human inherited hyperaminoaciduria that seems to result from an impairment of a system y+-like activity (497), is discussed in section III.

Two excellent recent reviews described functional and structural data as well as available data on the regulation of the expression of CAT-1 and CAT-2/2a transporters (105, 336).

1. Tissue expression

The tissue distribution of CAT genes has been examined by Northern blot analysis (for mCAT-1; Refs. 9, 242, 281; for hCAT-4, G. Sebastio, personal communication) and by RT-PCR (specific detection of mCAT-2 and mCAT-2a; Refs. 151, 336). Tissue distribution and transcript size of these genes are indicated in Table 3. Semi-quantitative data for the murine genes were summarized by MacLeod and Kakuda (336); all tissues or cell types examined express at least one of the mCAT genes, and in some tissues, both genes (CAT-1 and CAT-2) are expressed. Liver is the only tissue that expresses only mCAT-2a and not mCAT-1 or mCAT-2. Kidney, small intestine, resident macrophages and quiescent splenocytes, and T cells only express the mCAT-1 gene but neither of the two mCAT-2 splice variants. Upon activation, these cell types express the mCAT-2 variant. The rat and mouse CAT-3 gene is expressed specifically in brain, as a transcript of ~3.4 kb (219, 229). The human CAT-4 gene is expressed mainly in pancreas, skeletal muscle, heart, and placenta, and brain, lung, liver, and kidney show a faint band in Northern analysis (Sebastio, personal communication).

 
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  		TABLE 3.   Tissue distribution and transport characteristics of expressed CAT transporters

The tissue and subcellular distribution of CAT transporter isoforms is less known than their transcript distributions. Expression of mCAT-1 protein has been studied by exploiting its function as a viral receptor (infectivity or viral glycoprotein gp70 binding assay; Refs. 108, 281, 590, 610) and by using anti-mCAT-1 antisera (106, 108, 605). Recently, approaches to the detection of mCAT-1 based on its function as a viral receptor have been validated in null knockout CAT-1 mice (419); primary embryo fibroblasts from these mice were completely resistant to ecotropic retrovirus infection (i.e., mCAT-1 is the sole receptor for ecotropic murine leukemia virus). The lack of constitutive expression of CAT-1 in human, murine, and rat liver has been demonstrated by virus infectability studies (610) and immunofluorescence studies (605). These strategies served to demonstrate the induction of surface expression of CAT-1 in the murine liver after partial hepatectomy and after insulin and dexamethasone treatment (610).

Antibodies directed against the COOH terminus of CAT-2a, which do not distinguish between CAT-2 and CAT-2a, detected a peptide band of 70-80 kDa in liver (106). Very recently, MacLeod's and Closs' groups obtained antibodies capable of distinguishing between CAT-2 and CAT-2a. This is not an easy task, since the main difference between the two mCAT-2 splice variants is an eight-amino acid segment with five substitutions (see Fig. 2), but full reports on this issue have not yet appeared (C. MacLeod and E. Closs, personal communication). Therefore, confirmation, at the protein level, of the tissue distribution and regulation of the expression of the CAT-2 isoforms is expected to be reported soon. No antibodies have been reported against the rodent CAT-3 or the human CAT-4 proteins.

The specific tissue distribution and regulation of the expression of the different CAT transporter isoforms (see sect. IIA5) suggests a high level of regulation of the expression of the corresponding gene promoters and splice variants (336). MacLeods's (151) group has addressed this question for the mCAT-2 gene. The promoter region of mCAT-2 is extremely complex, with several 5'-untranslated regions (UTR) expanded in a genomic region of 19 kb from the first 5'-coding exon. For more detailed information, the reader is directed to the review by MacLeod and Kakuda (336). MacLeod's lab (151) described five exon 1 isoforms (1a, 1b, 1b/1c, 1c, and 1d) that splice into a common sequence 16 bp 5' of the AUG start methionine codon (151). Kavanaugh et al. (267) described from a liver clone a putative complex additional 5'-UTR (named 1e) of 515 bp, with six initiation and termination codons that precede the translation start codon, which is subjected to posttranscriptional regulation. Promoter 1a (at the far end of the complex promoter region) predominates over the others in every cell type and tissue examined (336). The promoter of exon 1a is a TATA-less one with staggered initiation, GC rich, and with several SP1 and CAC boxes. Liver (where only the mCAT-2a variant is expressed) and activated macrophages (where only the mCAT-2a variant is expressed) use promoter 1a exclusively (151); this demonstrates that promoter usage does not dictate the splicing events that render the mCAT-2 and mCAT-2a splice variants (336).

Posttranscriptional regulation of CAT-1 gene expression has been reported in liver regeneration (25), a model that induces system y+/CAT-1 isoform expression (see sect. IIA5). This study shows that accumulation of CAT-1 mRNA in the regenerating rat liver is due to posttranscriptional regulation, and there is no increased transcriptional activity (run-on experiments) of the gene; this posttranscriptional regulation is sensitive to cycloheximide. In the rat, CAT-1 produces two transcripts (7.9 and 3.4 kb in length), which represent alternative polyadenylation signal usage (the long transcript uses a consensus polyadenylation sequence, whereas the shorter uses a noncanonical signal); both transcripts accumulate in the regenerating liver. The specific 3'-UTR of the long transcript contains destabilizing AU-rich sequences that are associated with a shorter half-life (90 and 250 min for the long and short trancripts, respectively); interestingly, the longer transcript accumulates to higher levels than the shorter transcript. All this suggests that protein factors control the stability of CAT-1 mRNA through the long-specific 3'-UTR and through common sequences for both transcripts. In fact, cycloheximide administered in vivo to control rats upregulates the levels of the long transcript in several tissues but unfortunately not in liver, suggesting tissue-specific regulation of the half-life of CAT-1 transcripts. Similarly, in rat hepatoma FTO2B cells, where CAT-1 expression decreases with confluency, the relative abundance of the two transcripts also varies with confluency; the shorter transcript decreases faster than the longer (610). All this suggests that the 3'-UTR sequences of CAT-1 transcripts may be involved in the regulation of polyadenylation and/or stability of the transcripts (25). Unfortunately, in these studies showing differential expression of the two CAT-1 transcripts, no attempt was made to correlate transcript levels and CAT-1 protein abundance to assess translation efficiency. As an additional regulation mechanism of CAT-1 gene expression, in analogy with CAT-2 gene, these authors quoted unpublished results that suggest multiple promoter usage for the rat CAT-1 gene, but no report or confirmation of this is yet available.

To our knowledge, information on the promoter regulation of CAT genes to explain the modulation of CAT-1 and CAT-2 expression (see sect. IIA5) has not been reported.

2. Transport properties of CAT transporters

The characteristics of the amino acid transport acitivities elicited by the murine CAT transporters mCAT-1, mCAT-2, and mCAT-2a have been studied in Xenopus oocytes and are summarized in Table 3.

1) There is sodium-independent transport of cationic amino acids (e.g., L-arginine, L-lysine, and L-ornithine) with high affinity (Km in the micromolar range) by mCAT-1 and mCAT-2 (106, 108, 242, 267, 281, 590) and with low affinity (Km in the millimolar range for L-arginine) by mCAT-2a (106, 267). It is worth mentioning that Km values for L-ornithine influx are higher than those for L-arginine and L-lysine via mCAT-2, suggesting true differences in the extracellular recognition of these substrates (105). As shown in Table 3, there are discrepancies between labs as to the Km values of cationic amino acids in mCAT-1 and mCAT-2 transporters. Transport of cationic amino acids via mCAT-1 is voltage dependent; hyperpolarization increases the Vmax and decreases the apparent Km for influx (the reverse is true for efflux) (264). Closs (105) argued that differences of oocyte membrane potential in different labs and experimental conditions could underlie the variation reported for Km values.

2) For mCAT-1, mCAT-2, and mCAT-2a (242, 267, 590), the transport expressed has been shown to be electrogenic (positive charge follows the cationic amino acid flux) and stereospecific (i.e., Km in the millimolar range for D-cationic amino acids).

3) mCAT-1, mCAT-2, and mCAT-2a present trans-stimulation of arginine uptake, with mCAT-1 being more sensitive to this phenomenon (106, 108). mCAT-2a transport activity is largely independent of trans-side substrate (Table 3) (106, 108, 267).

4) Electrophysiological studies (242, 590) showed that mCAT-1 and mCAT-2 transport the zwitterionic amino acids homoserine and cysteine only in the presence of sodium (242, 590). Expression of low-affinity histidine uptake is also elicited by mCAT-1 and mCAT-2; for mCAT-1, it has been shown to be partially dependent on the presence of cis-sodium. At low pH, when histidine is protonated, this amino acid becomes a better substrate, demonstrating selectivity of CAT transporters for dibasic amino acids. For mCAT-2a (106), transport of zwitterionic amino acids in the presence of sodium has not been observed, although in these studies transport activity was measured by radioactive amino acid uptake, a less sensitive method than electrophysiological measurements.

At present, there are no data available on the amino acid transport activity expressed by hCAT-4 (but expression of hCAT-4 in oocytes resulted in increased L-arginine uptake; Sebastio, personal communication), and there are two reports on rat and mouse CAT-3 (Table 3). Transient expression of rCAT-3 in COS-7 cells resulted in sodium- and chloride-independent transport of radiolabeled L-arginine with an apparent Km of ~100 µM, inhibitable by cationic amino acids and dependent on membrane potential, as expected for a cationic amino acid transporter (219). Expression of mCAT-3 resulted in high-affinity, sodium-independent transport of dibasic amino acid, which shows trans-stimulation (229). Therefore, CAT-3 together with CAT-1 and CAT-2 transporters are high-affinity cationic amino acid transporters in contrast to the CAT-2a isoform.

It is worth mentioning that the proposed channel mode of action described for the sodium-dependent amino acid transporters, like members of the superfamilies of neurotransmitters and of excitatory amino acid transporters (see the corresponding sections), has not been described for the CAT transporters. It is interesting that these transporters and the proteins rBAT and 4F2hc, which essentially do not mediate sodium-dependent transport, do not seem to have a channel mode of action (90, 110).

Closs et al. (108) obtained surprising data on the accumulation capacity of mCAT-1, -2 and -2a transporters in oocytes at a nonphysiological extracellular concentration of 10 mM L-arginine. Incubation of oocytes in a high L-arginine concentration (10 mM) for 6 h, assuming an oocyte space distribution of ~180 nl/oocyte (90), leads to 0.6-fold accumulation in mCAT-1-expressing oocytes, 1.4-fold in mCAT-2-expressing oocytes, and 6-fold in mCAT-2a-expressing oocytes. These differences have been interpreted as the consequence of an apparent intracellular substrate affinity of mCAT-2a smaller than that of mCAT-1 and mCAT-2 (105). In our opinion, thermodynamic gradients are unlikely to be the result of substrate affinity differences. The regular oocyte membrane potential (-30 to -50 mV) is valid for an accumulation gradient of a positive charged substrate (i.e., L-arginine) of six- to eightfold. Interestingly, accumulation of 10 mM L-arginine in mCAT-2a-expressing oocytes from a sodium-free medium tends to this value (108). Why do mCAT-1 and mCAT-2 not reach the same accumulation gradient? In the experiments by Closs et al. (108), the membrane potential was not clamped, and therefore, the impact of the high L-arginine flux (10 mM extracellular concentration) was not controlled. Additional work at different extracellular substrate concentrations and at constant membrane potentials is needed to characterize the accumulation capacity of these transporters and the transport mechanisms underlying any possible difference between them.

For the human CAT-1, -2, and -2a counterparts, as for the mouse analogs, oocyte expression showed cationic amino acid transport of high affinity that was sensitive to trans-stimulation for hCAT-1 and hCAT-2 and cationic amino acid transport of low-affinity that was only slightly sensitive to trans-stimulation for hCAT-2a (unpublished data quoted in Ref. 105).

On the basis of all these characteristics (transport properties and tissue distribution), these cDNA (CAT-1, CAT-2, CAT-2a, and CAT-3) have been attributed to system y+ and its variants (242, 281, 590). In summary, transport activity elicited by mCAT-1, mCAT-2, and rCAT-3 expression is similar but with subtle differences (105, 219): sodium-independent high-affinity transport for cationic amino acids, with a slightly higher apparent affinity for mCAT-1, which is more sensitive to trans-stimulation. No data are reported on trans-stimulation via CAT-3. The transport properties and tissue distribution of CAT-1, CAT-2, and CAT-3 are consistent with subtle variants of system y+ (reviewed in Refs. 126, 600), the common cationic amino acid transport activity of mammalian cells. Most probably, CAT-2a represents a low-affinity liver variant of y+ activity. White and Christensen (601) described a low-affinity transport of L-arginine in primary hepatocytes not subjected to trans-stimulation and concluded that the classical y+ activity was absent or altered in hepatocytes. However, Van Winkle (574) suggested that the transport activities expressed by CAT-1 and CAT-2 could fit those of systems b+. Van Winkle et al. (579) also reassessed mCAT kinetic data, suggesting the presence of both a high- and a low-affinity component for each protein (mainly for mCAT-2a) when expressed in oocytes. At present, it is not clear whether this complex kinetic behavior represents an artifact of the expression model, different conformation or oligomeric states, or interaction with endogenous proteins. A more careful characterization of these amino acid transport activities based on inhibition by amino acids and analogs is needed to clarify this issue. Similarly, cell knockout or antisense experiments, like those reported for system bo,+-like in opossum kidney (OK) cells (374), would clarify the contribution of CAT transporters to y+/b+ transport activity in cells. In this sense, uptake studies in cells derived from the null knockout CAT-1 mice (419) may help to clarify this issue.

3. Protein structure of CAT transporters

Structural information on CAT transporters is scarce. All CAT transporters identified lack a characteristic signal peptide, and therefore, the NH2 terminus is considered to be cytoplasmic (see Refs. 105 and 336 for review and Ref. 219 for rCAT-3). Most of the additional information available has been obtained from mCAT-1, and it has been extrapolated to CAT-2 and CAT-3 transporters since they show almost identical hydrophobicity profiles (105, 219). These profiles initially suggested two membrane topology models for CAT transporters: 12 TM (according to MacLeod's and Saier's groups) or 14 TM (according to Cunningham's group) (9, 106, 334, 448). The two models differ in the middle TM domains (TM domains VII and X of the 14-TM model are considered to be intracellular in the 12-TM model; see Fig. 2). The 12-TM model is supported by the fact that CAT transporters belong to the APC transporter superfamily of yeast, fungi, and eubacteria, which presumably contain 12 TM domains (448), whereas the proposed membrane topology of the first 8 TM domains of the homologous yeast and fungi permeases argue in favor of the 14-TM model (105, 508). Mutational analysis showed that the viral binding site of mCAT-1 (see Fig. 2) is located between TM V and VI, confirming the extracellular location of extracellular loop (EL) 3 in both models (8). Evidence in favor of the 14-TM model has been obtained: 1) antibodies directed against peptides of the EL3 and EL4 loops of mCAT-1 in the 14-TM model immunostained nonpermeabilized cells (605). This confirmed the extracellular location of these protein regions; the 12-TM model predicts an intracellular location for the EL4 protein region. 2) The glycosylation of CAT transporters has been demonstrated by endoglycosidase F (endo F) treatment of immunodetected CAT-1 (i.e., antiserum raised against the COOH terminus of murine CAT-1) and CAT-2/CAT-2a (i.e., antiserum raised against COOH terminus of murine CAT-2a; a region that is identical to CAT-2) from mammalian cells or expressed in oocytes. These studies showed a broad glycosylated moiety of 3-9 kDa for mCAT-1 and mCAT-2a transporters (106, 280). Mutation in mCAT-1 of the two putative N-glycosylation sites Asn-223 and Asn-229 to histidine, conserved in all CAT transporters characterized (Fig. 1), results in a protein with identical SDS-PAGE mobility to the endo F-treated wild-type mCAT-1; mutation of either Asn residue results in an intermediate mobility. The 12-TM model predicts an additional, unconserved extracellular N-glycosylation site in mCAT-1 (Asn-373, located in the intracellular IL4 of the 14-TM model). Mutation of Asn-373 to histidine does not affect glycosylation of mCAT-1. These studies (280) demonstrated that Asn-223 and Asn-229 are the glycosylated residues of mCAT-1, and therefore extracellular, like the loop EL3 in the 14-TM model (Fig. 2), and that Asn-373 (in the IL4 loop of the 14-TM model) might not be located extracellularly, which thus favors the 14-TM model.

To our knowledge, extensive studies in search of evidence for the 14-TM model, like those performed with the GABA transporter GAT1 (38), the glycine transporter GLYT1 (401), and glutamate transporters (585, 498), have not been reported.

4. Structure-function relationship

Swapping chimeras with the divergent amino acid segment of CAT transporters, mutational analysis and studies related to the interaction with the murine ecotropic leukemia virus provided the core of our knowledge of the structure-function relationship for CAT transporters.

The apparent substrate affinity, maximum transport rate, trans-stimulation, and accumulation capacity are distinctive features of mCAT-2 and mCAT-2a (see Table 2, CATS). This suggests that these differential transport capacities are determined by the variant exon coding for the 41- to 42-amino acid residue divergent segment of the two proteins (see Fig. 2). Closs et al. (108) performed elegant studies, in which chimeric transporters with the backbone of mCAT-1 were completed with the divergent domain of mCAT-2 and mCAT-2a, and the backbone of mCAT-2 was completed with the corresponding domain of mCAT-1 (see Fig. 2). The transport characteristics (apparent Km, Vmax, trans-stimulation, and accumulation of L-arginine) of these chimeras expressed in oocytes are similar to those of the divergent region. Interestingly, the recently cloned rat CAT-3 expresses high-affinity (Km ~100 µM) L-arginine uptake in COS-7 cells, and its divergent domain is more similar to that of CAT-1 and CAT-2 than to that of CAT-2a (219) (see Fig. 2). These data suggest that the divergent protein domain of CAT transporters has an impact on all transport properties, and therefore, it might have a role in substrate recognition, turnover number, and the translocation mechanism. As indicated in Fig. 2, the divergent domain corresponds to the intracellular loop IL4 (including a few amino acid residues of TM domains VIII and IX) in the 14-TM domain model of CAT transporters.

Residues involved in the reported mutational analyses of mCAT-1 are indicated in the homologous position in the mCAT-2 protein model depicted in Figure 2. N-glycosylation is not required for transport function of mCAT-1 (280); the unglycosylated mutant (double Asn to His mutation at positions 223 and 229: see Fig. 1) expresses an unaffected transport activity in oocytes. The glutamate residue at position 107 of mCAT-1 is conserved in the TM domain III of all known CAT transporter sequences (see Figs. 1 and 2) and also in the yeast transporters for arginine, histidine, and choline of the APC family (589). This residue is required for transport activity in mCAT-1 protein expressed in mink CCL64 lung fibroblasts (589). Substitution by aspartate led to a loss of transport activity; interestingly, substitution by the uncharged glutamine residue did not affect transport activity (data by Kim and Cunningham quoted in Ref. 105). All these substitutions led to mCAT-1 protein expressed in the plasma membrane of the transfected cells as demonstrated by its role as a virus receptor (infectivity and viral glycoprotein gp70 binding). All this suggests that the carbon backbone size but not the negative charge of residue glutamate 107 of mCAT-1 determines transport function for the CAT transporters.

Meruelo and co-workers (621) and Cunninngham and co-workers (8) identified by domain swapping and mutational analyses the sequence NVKYGE (amino acid residues 232-237 in mCAT-1) within EL3 (see the corresponding position of this protein segment in Fig. 2) as essential for virus envelope binding and infection; swapping the above-mentioned sequence into the human CAT-1 conferred infectivity and virus binding (8). This sequence is also present in the rat CAT-1 counterpart (610), but not in hCAT-1 or the known CAT-2 proteins (see Figs. 1 and 2). Interestingly, both mCAT-1 and rat CAT-1 serve as a receptor for the virus. Detailed description of the amino acid residues within the EL3 loop required for binding of the viral protein envelope gp70 and permissivity to infection (8, 267) has been reviewed by Closs (105). It is worth mentioning that none of the mutations examined to determine viral interaction with CAT transporters affected their transport activity. In contrast, interaction of mCAT-1 with the virus reduces its transport activity. Coexpression of mCAT-1 and glycoprotein gp70 resulted in a specific reduction of mCAT-1 glycosylation and transport activity; a decrease in transport activity also occurs with the unglycosylated double Asn to His mutant at residues 223 and 229 (280). Binding of glycoprotein gp70 to the transfected mCAT-1 results in noncompetitive inhibition of amino acid import via the murine CAT-1 with no effect on amino acid export (588). The effects of gp70 on transport kinetics led the authors to suggest that gp70 binding represents a steric hindrance that slows the rate-limiting step of the amino acid import cycle, a conformational transition of the empty transporter in which the binding site moves from the inside back to the outside of the cell, and that gp70 has no effect on the rate-limiting step of the amino acid export cycle. A similar mobile carrier hypothesis has been suggested for the two-directional operation of the system y+ (600). The above-mentioned data suggest that amino acid transport and virus receptor functions may be coupled; conformational changes of the transporter may lead to membrane fusion of virus and host cell (105). Interestingly, the transport defective mCAT-1 mutant glu107asp mediates binding of glycoprotein gp70 and virus infection (589). This suggests that transport and receptor function may be uncoupled, but this is still an open question, since conformational changes for the transport-defective mutant have not been ruled out.

5. Physiological role of CAT transporters

An intriguing question is why there is such a variety of CAT transporters in mammalian cells. Cationic amino acids are needed for protein synthesis, urea synthesis (arginine), and as precursors of bioactive molecules (arginine and ornithine are substrates for the synthesis of urea and nitric oxide as well as polyamines, respectively). Then, what does each CAT transporter isoform contribute to the supply of substrates for these purposes? The contribution of other protein structures to cationic amino acid transport, like rBAT (system bo,+-like) and 4F2hc (system y+L-like) are discussed in section IID. The nearly ubiquitous CAT-1 isoform most probably corresponds, as discussed before, to the classical system y+, a high-affinity and electrogenic cationic amino acid transport system that allows accumulation of these substrates within the cells for general metabolic purposes. Consistent with this, the null knockout CAT-1 mice are smaller (limited accretion) at birth (419). In this sense, at a physiological extracellular concentration of L-arginine (50 µM), a high expression level of mCAT-1 in oocytes allows the maintenance of an L-arginine gradient across the plasma membrane of ~19-fold (90).

The known examples of upregulation of CAT-1 transporter expression favor this general role of the classical system y+/CAT-1 isoform. The expression of this gene is enhanced in proliferating cells (e.g., T and B lymphocytes activated by concanavalin A and bacterial lipopolysaccharide, rapidly growing cells infected with Friend leukemia virus, and a variety of tumor cells of different origin) (620). In liver regeneration, CAT-1 expression (both mRNA and protein) is induced a few hours after hepatectomy (25, 610). Recently, Hatzoglou's group (25) showed that CAT-1 could be considered as a delayed early growth response gene in the regenerating liver that requires protein synthesis for its upregulation. In keeping with this, ecotropic retroviruses infect hepatocytes during fetal development and liver regeneration, but not in adult hepatocytes (198). This supports a role of CAT-1 transporter in accretion, and as discussed by Wu et al. (610), a role of this transporter in the supply of ornithine for polyamine synthesis. Interestingly, the key enzyme in polyamine synthesis, ornithine decarboxylase, peaks during the G1 phase (145), and ornithine levels rise after partial hepatectomy (149). In addition to proliferation, hormone treatment (insulin and dexamethasone) also induces mCAT-1 expression in liver (610). A recent report also links CAT-1 expression with cell proliferation. Perkins et al. (419) reported that the homozygous null knockout CAT-1 mice develop anemia and die after birth. Erythroid maturation is defective in these mice because of a specific defect in cell proliferation and/or differentiation. In addition, this suggests that CAT-1 transporter is the main contributor to the cationic amino acid supply to erythroid progenitor cells. The specific contribution of CAT-1 transporter to the intracellular accumulation of cationic amino acids in those cells that express additional CAT isoforms (e.g., in brain, heart, skeletal muscle, uterus, ovary, testis, and placenta) is difficult to assess by indirect determinations, like transcripts and protein levels (see below). Specific knockout and antisense experiments are needed to delineate the contribution of each CAT transporter to the macroscopic amino acid flux through the plasma membrane of the cells.

The low-affinity high-capacity transport properties, the accumulation capacity through the plasma membrane at high extracellular substrate concentrations, and the exclusive expression of CAT-2a isoform mRNA, but not CAT-1 (both protein and mRNA) or CAT-2 (mRNA) isoforms, in liver have been envisaged as constituting a kinetic barrier between the hepatic urea cycle and extracellular arginine (105). Furthermore, on the basis of the low intracellular concentration of arginine in liver, it is unlikely that this amino acid is released through the activity of CAT-2. All this is consistent with the lack of activity of the classical high-affinity system y+ in the hepatocyte plasma membranes, which protects extracellular L-arginine from hydrolysis by hepatic arginase (600, 601). The hepatocyte CAT-2a transporter would allow rapid accumulation of cationic amino acids only at high plasma concentrations, leaving sufficient substrates in circulation for cells expressing the high-affinity CAT isoforms (105). In keeping with this, expression of CAT-1 isoform occurs in liver when the urea-cycle enzymes are downregulated (e.g., liver regeneration, insulin treatment, low-protein diet) (610). Interestingly, stress conditions (partial hepatectomy, surgical trauma, and fasting) upregulate mCAT-2a in skeletal muscle (K. D. Finley, quoted in Ref. 336); the CAT-2/-2a isoforms are prevalent in this tissue (242). Some of these stress situations have a muscle proteolytic state in common (309, 324). The possible physiological role of CAT-2a upregulation induced by stress conditions such as fasting in skeletal muscle is far from understood. MacLeod and Kakuda (336) suggested that this is a mechanism to prevent depletion of those amino acids from the tissue with active proteolysis. In this regard, it should be mentioned that the rate of release of lysine or arginine in the rat perfused hindquarter preparation is not modified in response to 48 h of fasting (464). Brief starvation in humans has been reported to enhance the release of lysine, but not of arginine, from skeletal muscle (429). Studies should be performed to determine the role of CAT-1, CAT-2a, and CAT-4 in the metabolism of cationic amino acids in skeletal muscle.

Ashcroft and co-workers (504) studied the transport mechanisms involved in the stimulation of insulin secretion by L-arginine in mouse pancreatic beta -cells. This work suggests that L-arginine raises the intracellular concentration of Ca2+ and stimulates insulin secretion as a consequence of its electrogenic transport into this cells. The expression of mCAT-2 and mCAT-2a in beta -cells was demonstrated by RT-PCR. L-Arginine produced a dose-dependent increase in the intracellular concentration of calcium, which suggests that the low-affinity mCAT-2a is the cationic amino acid transporter responsible for the secretagogue action of this amino acid. Specific mCAT-2a knockout experiments in beta -cells or in the whole animal are needed to demonstrate the role of mCAT-2a in the insulin secretagogue action of L-arginine.

The CAT-1 mRNA is constitutively expressed in mature resting and activated T cells and splenocytes and resident macrophages (242, 336). Activation of these cells mainly induces the CAT-2 transporter isoform. SL12 thymoma cell clones, a model system of thymocyte differentiation, show developmental regulation of mCAT-2 during thymocyte maturation (602). The mCAT-2 gene is downregulated in normal and mature thymocytes until it is activated by mitogens or antigens (151, 242, 334). Periphera