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Originally published In Press as doi:10.1074/jbc.M611520200 on May 7, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19788-19798, July 6, 2007
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Distinct Sensor Pathways in the Hierarchical Control of SNAT2, a Putative Amino Acid Transceptor, by Amino Acid Availability*Formula

Russell Hyde, Emma L. Cwiklinski, Katrina MacAulay, Peter M. Taylor, and Harinder S. Hundal1

From the Division of Molecular Physiology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom

Received for publication, December 15, 2006 , and in revised form, April 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Amino acids (AAs)2 act through the coordination of several signaling pathways to modulate distinct, albeit inter-related, processes (1-3), including protein synthesis and turnover, hormone action and release, and the synthesis, transport, and metabolism of AAs. The nutrient sensors that influence these processes are poorly defined in higher eukaryotes, so, in an attempt to clarify AA sensing in mammalian cells, the molecular regulation of a paradigm AA-sensitive process (the System A amino acid transporter) has been investigated.

Sodium-dependent neutral amino acid transporter 2 (SNAT2, encoded by the gene SLC38A2) exhibits functional and regulatory properties of the classically defined System A transporter (4). Hence, stimulation of System A by AA deprivation (aka adaptive regulation) or insulin occurs concurrent with increased SNAT2 expression (5) or the plasma membrane recruitment of SNAT2 (6), respectively. Palii et al. (7) isolated a tripartite AA responsive domain in the first intron of SLC38A2 that is required for increasing SLC38A2 mRNA during nutrient stress. Pharmacological and genetic interventions have implicated the classical extracellular signal-regulated kinase (ERK) and stress-activated Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) in the adaptive regulation of System A (8, 9) in certain cell types, although nutrient signaling loci upstream of MAPKs have not been identified to date.

System A substrates, including the synthetic AA analogue {alpha}-methylaminoisobutyric acid (Me-AIB), suppress the increase in SNAT2 expression that occurs in AA-starved cells (9-11). Hence, the concept that SNAT2 activity may regulate transporter expression has been developed and both direct and indirect mechanisms of transport-dependent sensing have been proposed (1). SNAT2 may function as a hybrid transporter-receptor (transceptor) whereby structural changes during the transport cycle may be transduced to signaling pathways; such AA transceptors are well documented in Saccharomyces cerevisiae (12, 13) but have yet to be verified in mammalian systems. However, this transceptor model cannot completely account for the control of System A/SNAT2, because AAs that are not substrates for this transporter (Trp, Tyr, Phe) also repress System A (14).

Here, AA deprivation is shown to stimulate functional expression of SNAT2 in rat L6 myotubes and HeLa cells through transcriptional induction and protein stabilization. The AA-dependent control of the latter process is conferred by a region in the N terminus of SNAT2. Inhibitors of JNK impair adaptive regulation and the AAs shown to regulate SNAT2 expression are either capable of restraining JNK phosphorylation and/or interacting with SNAT2. By using a shRNA approach it is demonstrated that SNAT2 functions as a mammalian AA transceptor, which acts in an autoregulatory gene expression pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Culture media ({alpha}-minimal essential medium, {alpha}-MEM, and Dulbeccos MEM), fetal calf serum and antibiotic/antimycotic solution were from Invitrogen (Paisley, UK). Radiochemicals were from PerkinElmer Life Sciences (Cambridge, UK). AAs and reagent grade chemicals were from Sigma-Aldrich (Poole, UK). Inhibitors were from Tocris (Bristol, UK) and CN Biosciences (Nottingham, UK).

Molecular Biology—Rat SNAT2 (15) and SNAT5 (16) cDNAs were placed in pcDNA6 (Invitrogen) following PCR mutagenesis to introduce a C-terminal V5-His6 tag. The SNAT2-SNAT5 NcoI chimera was generated in pCR2.1 and subcloned into pcDNA6. The rat SNAT2 promoter was cloned from L6 DNA and subcloned into pC3luc (see supplementary data). SNAT2 shRNAs were expressed from psiStrike (Promega) (Sequences: shSNAT2, ACCGATGAAC GTGTCCAAGA TTAAGTTCTC TAATCTTGGA CACGTTCATC TTTTTC; shSCRAMBLE, ACCGCTGGTT CAAACGTTAA GAAAGTTCTC TTCTTAACGT TTGAACCAGC TTTTTC).

Cell Culture—L6 cells were grown and cultured to the stage of myotubes as described (6). HEK 293 and HeLa cells were grown in Dulbecco's modified Eagle's medium/10% fetal calf serum/1% antibiotics. For experiments, cells were incubated in Earls' Balanced Salt Solution (EBSS) supplemented with an AA mix at 1x physiological concentration (5) for 1 h, rinsed, and incubated in the appropriate buffer for cell stimulation. Monolayers were rinsed prior to lysis or cell membrane isolation (17). L6/HeLa cells transfected with pC3luc constructs were incubated in growth medium supplemented with 0.8 mg/ml G418 (Melford, Suffolk, UK). HEK 293 and HeLa cells were transiently transfected with pCDNA6 or psiStrike constructs (10 µg DNA/10-cm plate) using calcium phosphate.

Solute Transport—Transport measurements were performed as previously described (17), with slight alterations. Following cellular stimulations, L6 cells were rinsed with Hepes-buffered saline (HBS), exposed to uptake solutions (typically 10 µM [14C]Me-AIB (1 µCi/ml) in HBS) for 10 min at room temperature, then rinsed 3x with 0.9% NaCl (4 °C) and lysed in 50 mM NaOH. Nonspecific radiotracer binding was assessed with 10 mM unlabeled Me-AIB (or by brief tracer exposure in kinetics experiments). For cis-inhibition experiments, the uptake solution was supplemented with unlabeled AAs as noted. For trans-inhibition experiments (described under supplementary data), L6 myotubes were preincubated in AA-free EBSS for 4 h and then exposed to 2 mM unlabeled AA for 20 min (at 37 °C). Me-AIB uptake was subsequently measured as above. Uptake was standardized against protein recovery, or is presented relative to a control value.

Immunoblotting—20-50 µg of cell protein was separated by SDS-PAGE as previously described (17). Polyvinylidene difluoride membranes were probed with signaling antibodies (New England Biolabs, Hitchin, UK), monoclonal antibodies against the Na/K-ATPase {alpha}1 subunit ({alpha}6F; 1/8000; DSHB, University of Iowa), the V5 epitope (1/5000; Invitrogen) or chicken anti-SNAT2 IgY (1/200, ~0.5 µg/ml; Antigen, FLLESNLGKYET).

Luciferase Activity—Luciferase assay was performed in 20 µg of cell lysate using the Luciferase Assay System (Promega, Southampton, UK; according to the manufacturer's protocol) in a TD20/20 luminometer (Turner BioSystems, Sunnyvale, CA). Luciferase activity in experimental samples was standardized relative to serum-starved, AA-supplemented cells.

Immunofluorescence—HEK 293 cells were grown on coverslips and transfected with pcDNA6-V5His-Version A (Empty Vector, Invitrogen), pcDNA6-LacZ-V5His or the pcDNA6-SNAT2-V5His construct. Cells were rinsed with PBS and fixed with paraformaldehyde (4%, w/v) in PBS for 30 min. Cells were rinsed twice with PBS and rehydrated in PBS/2% fish skin gelatin (w/v) for 30 min. Cells were then incubated in PBS in the presence or absence of 0.2% Triton X-100 for 2 min. Detergent was removed with four changes of PBS. Coverslips were incubated overnight with anti-V5 monoclonal antibodies (1/250, v/v) in PBS/2% fish skin gelatin, were subsequently rinsed with PBS, and were incubated with fluorescein-conjugated anti-mouse antibody in PBS/gelatin. The rinsed and mounted coverslips were visualized by fluorescence microscopy.

Statistical Analysis—One-way analysis of variance and non-linear regression were performed using GraphPad Prism 4 software and considered statistically significant at p < 0.05. For the non-linear regression analyses in Fig. 6, Equations 1 (inhibition) and 2 (repression) were used,

Formula(Eq.1)

Formula(Eq.2)

where the parameters ymax, ymin, and Kd are derived from the best-fit curve to the experimental data (y being transport rate/relative luciferase activity and x being inhibitor/repressor concentration) using GraphPad Prism 4.

Online Supplemental Material—Supplementary methods containing extended protocols (cloning, transfection, buffers, IgY generation, and purification) and additional data showing: (i) the time-dependent increase in System A activity during AA deprivation; (ii) the repression of System A activity by individual AAs; (iii) an alignment of the N termini of vertebrate SNAT2 orthologues; (iv) the lack of effect of CaR modulators on adaptive regulation of System A; (v) the effect of altered extracellular pH on System A transport and adaptive regulation; and (vi) a comparison of the ability of individual AAs to cis-inhibit and trans-inhibit System A are available online.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Amino Acid Specificity of System A Regulation—AA starvation of L6 myotubes induces a time-dependent increase in System A activity ((5), Fig. 1A, and supplemental Fig. S1) through a process dependent upon new protein synthesis (net induction in nutrient-deprived cells: 92.8 ± 32.3 pmol/min/mg; net induction in the presence of the translation inhibitor cycloheximide: 16.1 ± 3.8 pmol/min/mg; p < 0.05). The AAs that regulate System A activity were determined by providing individual AAs (at 2 mM) to otherwise AA-starved myotubes (Fig. 1A and supplemental Fig. S2). Consistent with the idea that the System A transporter might generate a repressive signal during the transport cycle, AAs shown to be the best substrates for SNAT2 (18) were generally the compounds with the greatest repressive effect, although aromatic AAs were also found to significantly repress System A induction (p < 0.05). Cis-inhibition experiments were used to demonstrate that although 2 mM Tyr impaired System A up-regulation, Tyr did not significantly interact with System A at this concentration (Fig. 1B). Nevertheless, (L)-Gln, Ala, and Me-AIB all interact with SNAT2 and the non-repressive AAs did not (Fig. 1B).

SNAT2 abundance increases in AA-deprived L6 myotubes (5). Hence, the effects of the above AAs on SNAT2 protein expression were assessed using chicken anti-SNAT2 antibodies (Fig. 1C). These antibodies were designed against a region in the cytoplasmic N-terminal domain of SNAT2 (FLLESNLGKYET) and bind to two broad bands ({approx}35 kDa and 55 kDa) that increase in response to AA deprivation. Given the co-regulation of these proteins we propose that the higher band is full-length SNAT2, and the lower band is a cleaved form of SNAT2. A cross-reactive band ({approx}75 kDa) is also observed; however, this protein is restricted to intracellular membranes (data not shown). Tyr, Ala, and (L)-Gln prevented the induction of SNAT2 protein and Me-AIB had a slight repressive effect after 4 h (Fig. 1C), which was almost complete after 6 h of incubation with Me-AIB. Amino acids that were non-repressive following4hof stimulation (Leu, Tyr, Arg) remained non-repressive even when incubated with cells for 6 h. Because Me-AIB had a modest repressive effect on SNAT2 expression, but a large effect on System A transport, the overall repressive effect of Me-AIB on System A activity may largely be due to trans-inhibition of plasma membrane SNAT2 by cytoplasmic Me-AIB (19). To test whether Me-AIB exerted a repressive effect strictly on SNAT2 gene expression, analysis of the SNAT2/SLC38A2 promoter was performed.


Figure 1
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  		FIGURE 1.
Regulation of System A by amino acids. A, L6 myotubes were incubated ± the 1x AA mix, or in EBSS + 2mM of the indicated AA for 4 h prior to measurement of 10 µM Me-AIB transport. B, 10 µM Me-AIB transport was measured either in the absence (Control) or in the presence of 2 mM unlabeled AA in AA-starved L6 cells. C, expression of SNAT2 and {alpha}1-Na-pump in L6 myotubes following 4 h (left panel) or 6 h (right panel) incubation ± AA. D, luciferase activity in stably transfected L6 cells exposed to EBSS ± AA for 7 h. Luciferase was driven by CMV, a SNAT2 promoter (-1 + 1) including the full 5'-UTR or the same promoter with a truncated UTR (-1 + 0.3), as illustrated. Abbreviations as defined in the text. Data are mean ± S.E. for 4-5 experiments. **, p < 0.01 versus AA-deprived (1A+D) or non-inhibited (B) values.

 
Amino Acids Control SNAT2 Transcription and Protein Stability—The SLC38A2 AA responsive domain isolated by Palii et al. contains three elements, a CAAT-box, an amino acid response element (AARE in Fig. 1D) and a purine-rich element (PuR in Fig. 1D), each of which contribute to SNAT2 expression during AA limitation (7). The rat SNAT2 promoter was cloned and two versions of this promoter, one (-1 + 1) containing the first intron of the gene and one (-1 + 0.3), which lacks the first intron and its associated nutrient responsive domain, were placed upstream of firefly luciferase (Fig. 1D). SNAT2-luciferase vectors were stably expressed in L6 cells. The inclusion of 1 kb of upstream sequence in both SNAT2-luciferase promoters produced a substantial basal level of transcription relative to CMV minimal promoter (Fig. 1D), as also seen with upstream regions of the mouse SNAT2 promoter (7). The SNAT2-1+1-luciferase transgene was significantly induced following7hofAA deprivation (Fig. 1D). SNAT2-luciferase induction was dependent upon the presence of intron 1, because SNAT2-1+0.3-luciferase activity was not stimulated by AA deprivation. The relative activation produced by the nutrient responsive domain of rat SNAT2 intron 1 in the absence or presence of AA is around 2.2 (Fig. 1D), which, given the short duration of AA starvation we used (7 h versus 10 or 12 h in other studies), is comparable to results reported elsewhere (~1.8-2.5 for mouse SNAT2 intron 1 (7); ~3 for human SNAT2 intron 1, (20). As a further negative control, a luciferase construct driven by the cytomegalovirus (CMV) promoter was also unaffected by nutrient availability. In the presence of the repressive AAs ((L)-Gln, Ala, Tyr, Me-AIB; 2 mM), SNAT2-1+1-luciferase activity was reduced to control (AA+) levels, whereas Arg, a non-repressive AA, did not affect expression of this construct.


Figure 2
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  		FIGURE 2.
SNAT2 protein turnover is controlled by amino acids. A, HeLa cells were transfected with pcDNA6-SNAT2V5, SNAT5V5, or an NcoI-SNAT2-SNAT5V5 chimera, and/or with psiStrike-driven SNAT2 shRNA (or scrambled control). Cells were incubated ± AA for 8 h and transgene expression determined by anti-V5 immunoblotting. The final panel shows a low exposure of the blot depicting SNAT5V5 expression in SNAT2 silenced cells. B, cartoon depiction of the 12-pass and 11-pass models for SNAT2 structure and the SNAT2/SNAT5 NcoI sites used in chimera generation. C, anti-V5 immunofluorescence in SNAT2-V5- and LacZ-V5-transfected HEK-293 cells. Cells in the lower panels were permeabilized with Triton X-100.

 
Given that nutrient-responsive transcriptional controls appear to impart only a modest stimulus for SNAT2 induction, we examined the possibility that additional levels of control contributed to transporter activation. Hatzoglou's group (20) has recently found an internal ribosome entry sequence (IRES) in the 5'-untranslated region (UTR) of the SLC38A2 gene; however, unlike in certain other transcripts where an IRES has been documented (e.g. CAT1 (21)), the SNAT2 IRES does not stimulate translation but permits uninterrupted transporter expression during nutrient stress, a condition where global translation initiation may be restricted.

We proposed that SNAT2 protein may be stabilized during nutrient stress and to test this we expressed a CMV-driven SNAT2 construct (containing a C-terminal V5-His6 epitope tag) in HeLa cells, since these are more readily transfected than L6 cells and in separate experiments (not shown) were found to also exhibit System A adaptation in a JNK-dependent manner. V5 immunoreactivity was observed in lysates of SNAT2V5-transfected HeLa cells with a banding pattern appropriate for the full-length SNAT2 glycoprotein (55-60 kDa). The SNAT2V5 signal was absent from cells which had been cotransfected with anti-SNAT2 shRNAs (but not from cells transfected with scrambled shRNA controls), thus validating that the protein bands observed are a specific consequence of SNAT2V5 expression. AA deprivation of the SNAT2V5-transfected HeLa cells for 8 h led to a marked increase in V5 immunoreactivity, implying an increased abundance of SNAT2V5 protein in these cells (Fig. 2A).

Our studies in L6 cells had shown that the CMV promoter used here was not sensitive to AA-deprivation (Fig. 1D) and to verify the specificity of this AA-dependent increase in SNAT2V5 protein, rat SNAT5 (a structurally related transporter from the SLC38 gene family) was V5-tagged and expressed in HeLa cells. Although analogous to the SNAT2V5 construct, an equivalent induction of SNAT5V5 under nutrient deprivation was not observed (Fig. 2A). Additionally, given that the two constructs were expressed from an identical transcriptional promoter (CMV), SNAT5V5 appears to be considerably more stable than SNAT2V5 in HeLa cells. Our interpretation of these data is that during AA-sufficiency SNAT2 protein is considerably less stable than during AA-restriction.

A corollary of the proposal that SNAT2 is stabilized during nutrient deprivation is that a particular region of the SNAT2 sequence may confer AA-regulated stability effects. It should therefore be possible to identify such regions through mutagenesis. The cytoplasmic domains of SNAT2 are the best candidate regions for associating with the proteolytic machinery; however, the structure of SNAT2 within the cell membrane is poorly characterized. Two topologies have been proposed which differ in the number of transmembrane domains (15, 18, 22), as illustrated in Fig. 2B. Both models predict a large intracellular N terminus, but the 12-pass model proposed by Sugawara et al. (22) differs from the 11-pass model of Yao et al. (15) and Reimer et al. (18) in placing the C terminus at an intracellular location. To help distinguish between the two models, SNAT2V5 was expressed in HEK 293 cells and the cells were subject to analysis by immunofluorescence (Fig. 2C). Unlike the cytoplasmically localized LacZV5 control, HEK293 cells transfected with SNAT2V5 did not require permeabilization with Triton X-100 to allow an appreciable signal to be observed in anti-V5 immunofluorescence. Hence, in SNAT2V5, the C-terminal V5 epitope is accessible from the extracellular domain, supporting the 11-pass topology of SNAT2.


Figure 3
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  		FIGURE 3.
Signal transduction and System A adaptive regulation. L6 myotubes were exposed to EBSS ± AA for 4 h (A) or the indicated time period (B). Incubations were performed with signaling inhibitors or vehicle (as stated in the text) prior to assessing 10 µM Me-AIB transport (A) or the phosphorylation status of several AA-responsive proteins (B). In A, the net induction of System A activity (VAA- - VAA+) is presented relative to System A induction in the presence of 2% Me2SO (v/v) vehicle. Data are mean ± S.E. for 3-13 experiments. */**, p < 0.05/p < 0.01. In B, certain cells were treated with tunicamycin (5 µg/ml) for 4 h as a control for eIF2{alpha} phosphorylation. Western blots of cell lysates were performed with JNK-P (1/400), SEK-P (1/400), ERK-P (1/1000), GSK3-P (1/1000), S6K-389-P (1/1000), eIF2{alpha}-P (1/400), or native JNK (1/1000) antibodies.

 
Sequence identity between SNAT proteins is highest within the transmembrane domains and lowest within the hydrophilic N terminus (see supplemental Fig. S3) and the large extracellular loop and consequently it is predicted that isoform-specific regulatory domains are present within the hydrophilic regions. To test whether the N terminus of SNAT2 might control AA-dependent stability, a CMV-driven chimera was generated between SNAT2 and SNAT5V5 using a conserved NcoI site that maps to the first extracellular loop of the two proteins (as illustrated, Fig. 2B). Only 3 nucleotides from the SNAT2 5'-UTR were present in this construct (to minimize the potential influence of the 5'-UTR in AA-dependent control). Upon transfection of HeLa cells, SNAT2-5V5 protein expression was comparable to that of SNAT2V5 and was increased upon AA deprivation (Fig. 2A). Hence, by grafting the SNAT2 N terminus onto SNAT5 we have rendered the stability of the SNAT5 protein sensitive to AA availability.

Signaling Pathways Mediating System A Adaptation—AA availability controls a variety of signaling pathways. To identify those pathways relevant to System A regulation, L6 myotubes were incubated with signal transduction inhibitors during our adaptation protocol prior to measuring Me-AIB transport (Fig. 3A). Inhibitors of the ERK pathway (PD-098059, 50 µM), the p38 MAPK pathway (SB-203580, 10 µM), or mTOR (rapamycin, 100 nM), neither mimicked (data not shown) nor impaired System A adaptation. In contrast, inhibitors of PI3K (wortmannin, 100 nM, and LY-294002, 50 µM) and JNK (SP-600125, 30 µM) significantly inhibited System A adaptation. 3-methyladenine (10 mM) inhibits class III PI3K isoforms (23) and has been shown to inhibit autophagic proteolysis in AA-starved mammalian cells. This compound did not affect System A adaptation, which argues against a role for class III PI3K in the nutritional regulation of System A.

AA withdrawal is shown to increase the phosphorylation (activation) of both ERK and JNK in L6 cells (Fig. 3B). Compared with previous studies (8, 9), wherein JNK and ERK signaling peaks within 30 min, MAPK activation was both delayed and prolonged in L6 cells, being apparent between 2 and 6 h of nutrient restriction. During AA deprivation, SEK1 (a MAPK kinase lying directly upstream of JNK) was rapidly phosphorylated, whereas the mTOR target p70-S6 kinase (S6K) was dephosphorylated and glycogen synthase kinase 3 (a downstream effector of class I PI3K signaling) was unaffected. The protein kinase GCN2 has been shown to phosphorylate the translation factor eIF2{alpha} during amino acid restriction (24); however, although eIF2{alpha} phosphorylation increased with prolonged incubation time, AA withdrawal did not affect this in L6 myotubes. Tunicamycin, which affects eIF2{alpha} independently of GCN2, did stimulate eIF2{alpha} phosphorylation in L6 myotubes (21).

Ligand Sensitivity of JNK Signaling—If a single AA sensor was responsible for restraining SNAT2 adaptation, then that same sensor should also restrain signaling through the pathways that induce SNAT2 expression. Data from ourselves (this report) and others (9) implicate JNK in this process. However, relative to the GCN2 and mTOR pathways, AA signaling via JNK is poorly documented. To address this, we assessed the ability of specific AAs to inhibit the JNK activation typically observed in AA-starved cells (Fig. 4). Of the AAs tested, (L)-Gln and Tyr (2 mM) substantially reduced JNK phosphorylation, while Ala (also Ser and Trp) had more modest inhibitory effects. (L)-Gln, Tyr, and Ala also repress SNAT2 expression (Fig. 1, C and D) and consequently inhibit System A adaptation (Fig. 1A). Leu, Arg, (D)-Gln, and Me-AIB did not affect JNK phosphorylation. ERK inactivation by AAs appeared to have identical ligand selectivity to that of JNK, whereas the sensitivity of the mTOR pathway (judged by S6K phosphorylation) is distinct from the MAPKs. A shared AA sensor may therefore regulate the two MAPKs, while separate sensor processes may control mTOR.


Figure 4
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  		FIGURE 4.
Amino acid specificity of cell signaling. A, L6 myotubes were incubated for 4 h in EBSS ± AA, or with 2 mM of the indicated AA. Western blots were performed as described in the legend to Fig. 3, plus, S6K 421/424-P (1/1000). B, quantitation of JNK phosphorylation for experiments of the type shown in A was performed using ImageJ software; densitometry data are normalized to native JNK abundance in each lane, setting the value -AA to 100%. Values are mean ± S.E. for 3-4 experiments.

 


Figure 5
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  		FIGURE 5.
SNAT2 expression level modulates SNAT2 promoter activity. HeLa cells stably expressing the pC3luc-SNAT2-1+1 luciferase construct were transiently transfected with pcDNA6-SNAT2V5 or psiStrike-SNAT2 shRNA constructs (and scrambled (shScram) controls). Cells were incubated ± AA for 8 h prior to lysis, and luciferase activity was assessed in 10 µg of protein lysate. Values are mean ± S.E. for 3-4 experiments.

 
The extracellular Ca receptor (CaR) functions as a sensor for large neutral AAs and AAs enhance the Ca2+ affinity of CaR (25). Given that aromatic AAs regulate JNK and are among the better ligands for CaR, we tested whether CaR contributed to System A adaptation by incubating L6 cells in the absence or in the presence of excess (10 mM) Ca2+ over a 4-h period of AA starvation (supplemental Fig. S4). CaR does not appear to control System A activity because the ability of a CaR agonist (2 mM Trp) to repress System A does not depend upon extracellular Ca2+. Furthermore, excess Ca2+ did not mimic the repressive effect of extracellular AAs and the alternative CaR ligands spermine (0.5 mM) and neomycin (120 µM) were also without effect. Nevertheless, preliminary data (not shown) places the MAPK regulating sensor at the plasma membrane, because (i) (D)-Tyr, which is poorly transported, regulates JNK; and (ii) an inhibitor of tyrosine transport (2-amino-2-norbane-carboxylic acid (BCH)) does not prevent the repressive effect of (L)-Tyr, but has no effect itself. The lack of repression by leucine or BCH appears to exclude the possibility that this sensor is a transporter for bulky hydrophobic/branched chain amino acids.

We have not found any AAs that inhibit JNK, which do not also repress System A. Despite this, inhibition of JNK signaling cannot completely account for the repression of System A, because repressive AAs exist which do not interfere with JNK; for example, Me-AIB inhibits the stimulation of SNAT2 expression in L6 cells (Fig. 1) and has also been shown to impair the induction of SNAT2 mRNA by three other groups (9-11). Given that Me-AIB does not affect JNK phosphorylation (Fig. 4A), we propose that a second sensor with distinct, although overlapping, AA sensitivity functions in parallel with the putative JNK-regulating AA sensor to control System A activity, namely SNAT2 itself.

SNAT2 as an Amino Acid Sensor: Molecular Analysis—Similarities between the AAs that regulate System A expression and that function as System A substrates have long been appreciated and led Gazzola et al. (14, 26) to propose that AA binding by System A generates a signal that impairs gene expression of the System A carrier. On this basis we reasoned that manipulating the expression of SNAT2 would affect nutrient sensing by the carrier.

HeLa cells were stably transfected with the SNAT2-1+1 luciferase construct and subsequently transiently transfected with the SNAT2V5 construct (as used in Fig. 2). Overexpression of SNAT2 in these cells resulted in a reduction in basal luciferase activity in AA-containing medium (Fig. 5). Consistent with this observation, knocking down SNAT2 expression using shRNA led to an enhanced activity of the luciferase reporter in the AA-fed state, whereas scrambled shRNA hairpins had no effect on the SNAT2 promoter. These data indicate the existence of a feedback mechanism that allows SNAT2 to contribute to the repression of its own promoter during AA sufficiency. It is also evident from Fig. 5 that the proportional increase in SNAT2-luciferase activity following AA withdrawal is broadly similar under all conditions studied (1.44 no DNA; 1.36 SNAT2V5; 1.31 shScramble; 1.55 shSNAT2), hence the activation of MAPK signaling and the relief of SNAT2-based repressive signaling in parallel may be required to permit adaptive induction of SNAT2 transcription. Evidence for such convergence is seen in Fig. 1, where Tyr and Me-AIB inhibit SNAT2 luciferase to the same extent, despite acting on distinct signaling pathways.


Figure 6
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  		FIGURE 6.
Amino acid affinities for SNAT2 and for repression of transporter induction. Wild-type L6 cells were incubated4hin EBSS ± a range of tyrosine (Tyr), sarcosine (Sarc), or Me-AIB concentrations prior to lysis (A) or 10 µM Me-AIB transport assay (Repression; B and C). L6 cells transfected with SNAT2-1+1-luciferase were incubated in EBSS ± a range of Me-AIB concentrations for 7 h (D). A, JNK phosphorylation in L6 cells as a function of AA concentration. B and C, for cis-inhibition studies, AA-starved cells were incubated with 10 µM [14C]Me-AIB plus unlabeled Tyr/Sarcosine; Inhibition/Repression data are presented relative to non-inhibited transport (100%). D, luciferase activity is presented relative to AA + control; For MeAIB transport kinetics, AA-starved cells were incubated with [14C]Me-AIB tracer plus the indicated Me-AIB trace concentration. Data in B-D are mean ± S.E. for a minimum of three experiments and were analyzed by non-linear regression as described in the text (curves of best-fit are shown). In D, luciferase data were also modeled against Equation 2 with the Kd value set to 240 µM (dashed line). */**, p < 0.05/p < 0.01, relative to AA-starved control.

 
SNAT2 as an Amino Acid Sensor: Amino Acid Binding Kinetics—A further corollary of our transceptor hypothesis is that the affinity of SNAT2 for its AA substrates should determine the extent of AA-signaling by the carrier. Similarly, the extent of JNK signaling should echo the affinity of the AA ligand for the (putative) JNK-regulating receptor. Given that the two sensor/effector pathways are proposed to function in parallel, the sensor for which any given AA has the highest affinity will be that providing the first wave of repressive control toward SNAT2 expression as the concentration of that AA is increased. Several of the SNAT2 substrates studied were also able to inhibit JNK (Gln, Ala; Fig. 4A) and may control SNAT2 expression through a composite of their influences on SNAT2 and JNK. However, the signaling/sensing networks which contribute to SNAT2 regulation may be more readily dissected through studying compounds which target a single sensor.

At first glance, Me-AIB appears to be a good candidate for dose-response analysis of the SNAT2 transceptor. However, although Me-AIB does not inhibit JNK phosphorylation (even when provided at 10 mM, Fig. 6A) the compound is inappropriate for these studies since it trans-inhibits the System A carrier (supplemental Table S1). Trans-inhibition is a rapidly invoked kinetic effect of a subset of the System A substrates (Met, Me-AIB, Pro, AIB, Gln, His) and is most likely due to the cytoplasmic trapping of the System A carrier in an inwardly facing direction when presented with high intracellular AA concentration (19). Trans-inhibition is problematic for the current studies since, by knocking down the functional activity of the System A carrier, trans-inhibitory AAs effectively mask any repressive capacity that they may exhibit. In contrast to MeAIB, the alanine isomer sarcosine was found to cis-inhibit System A activity without displaying an appreciable level of trans-inhibition (supplemental Table S1). Furthermore, over a range of sarcosine concentrations (2-10 mM) the compound was without effect on JNK phosphorylation (Fig. 6A). We therefore used sarcosine as a relatively specific ligand for SNAT2-dependent AA signaling in this instance, hence, if our model is correct, the affinity of this compound for SNAT2 should be the sole determinant of its repressive capacity, Analysis of the dose-dependent effects of sarcosine support this proposal (KdRepression = 2.2 ± 2.0 mM; K Inhibitiond = 2.4 ± 0.6 mM, n = 5; Fig. 6C).

In contrast to the effects of sarcosine, Tyr (at 2 mM) was shown to repress System A without significantly interacting with the carrier (see Fig. 1). The same concentration of Tyr also reduces JNK phosphorylation (Fig. 4 and 6A). Hence, Tyr appears to have a higher affinity for the putative JNK-regulating receptor than for SNAT2. In Fig. 6B, non-linear regression was used to determine Kd values for repression of the adaptive response and inhibition of System A activity by Tyr (1.2 ± 0.3 mM and 19.4 ± 3.1 mM, respectively; mean ± S.E., n = 4). The concentration-curve for repression by Tyr thus better reflects the ability of Tyr to regulate JNK (Fig. 6A) than its ability to interact with SNAT2. Similar results were obtained with tryptophan (data not shown).

These data are in agreement with our suggestion that SNAT2 autoregulates its own expression in parallel with a sensor with distinct AA selectivity, which regulates JNK. Nonetheless, we wanted to validate that the effects of Me-AIB on SNAT2 gene expression tied together with the affinity of SNAT2 for Me-AIB, given that it was because of the regulatory effects of this non-metabolizable AA that the present study was initiated. To bypass problems of trans-inhibition with Me-AIB, an alternative approach to directly examine SNAT2 gene induction was necessary. Hence, L6 cells stably expressing the SNAT2-1+1-luciferase construct were incubated for 7 h in the presence of various concentrations of Me-AIB (10 µM to 10 mM), and cell lysates were subsequently assessed for luciferase activity (Fig. 6D). In parallel experiments the transport kinetics of the L6 System A carrier were determined. The affinity of the L6 System A carrier for Me-AIB (Km = 0.24 ± 0.06 mM) was of similar order to published values for rat SNAT2 (0.14-0.50 mM (4)). The luciferase activities were standardized against AA-fed cells and were analyzed by non-linear regression (as for the Sarc/Tyr repression studies above). The Kd value thus obtained for repression of the SNAT2-1+1-luciferase transgene by Me-AIB was 0.54 ± 0.39 mM, which is close to the Km for Me-AIB transport. This similarity between the dose-dependent effects of Me-AIB for transporter interaction and the regulation of gene expression is clear when comparing the luciferase data to a fitted line having a Kd value of 0.24 mM (dashed line in Fig. 6D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified two distinct nutrient sensing and signaling mechanisms which contribute to the repressive effects of individual AAs on the adaptive regulation of the System A (SNAT2) transporter: (i) A mechanism suppressing activation of the MAP kinase JNK by nutrient stress (responsive to several AAs, not all of which are SNAT2 substrates) and (ii) A JNK-independent mechanism, for SNAT2 substrates only, which is most prominent for substrates which do not also suppress JNK activation (e.g. sarcosine, MeAIB). The Kd by which these substrates repress the adaptive response is similar to the Km for their binding at the extracellular face of SNAT2, suggestive of a transceptor function for SNAT2.

Amino Acid-dependent Protein Kinase Signaling—The observation that aromatic AAs repress System A adaptation without interacting with the carrier itself (14) has been used as evidence against the proposition that the System A carrier has a function in AA sensing (26), Our data provide an explanation for this discrepancy by revealing an additional amino acid sensor upstream of JNK and ERK in L6 cells. This putative AA sensor (which is distinct from CaR) may also have a role in nutrient regulation of JNK/ERK-dependent processes outwith AA transport; e.g. AA deprivation stimulates autophagic proteolysis through an ERK-dependent pathway (27) and there is great similarity in the AA sensitivity of JNK/ERK signaling in our studies and early work studying the regulation of hepatic autophagy (28). The paradigm JNK substrate jun is known to bind the SNAT2 promoter (29). The reason why inhibition of the ERK pathway did not impair adaptive regulation in L6 cells, despite reproducible activation of ERK by AA withdrawal, may be either that a downstream component of the ERK pathway is absent (or non-responsive to ERK signaling) in L6 cells or that its effects are redundant.

The GCN2 signaling pathway is another candidate for mediating aspects of the transcriptional induction of SNAT2 (20, 29). In AA-deprived cells, uncharged tRNA accumulation activates GCN2, which phosphorylates the translation factor eIF2{alpha}, permitting translation of the transcription factor ATF4 (24), and the induction of nutrient-regulated genes such as asparagine synthetase (AS). ATF4 binds to the SNAT2 promoter during histidine deprivation (29, 30), and System A adaptation may be reduced (20) in cells where components of the GCN2 pathway are blocked. Nevertheless the GCN2 pathway does not appear to be an important mediator of the SNAT2 adaptive regulatory response in L6 muscle cells, at least during complete AA withdrawal, for several reasons: (i) in L6 cells there is no elevation of phospho-eIF2{alpha} within a time period (4 h; see Fig. 3B) sufficient to observe SNAT2 induction/System A activation (Fig. 1), nor is there any induction of System y+ activity (a process indicative of eIF2{alpha} phosphorylation in other cell types (e.g. see Ref. 31) as measured by [3H]Arg uptake; (ii) glucose deprivation (which independently mimics GCN2 signaling (32)) for 4 h does stimulate eIF2{alpha} phosphorylation, but did not promote an increase in System A activity in L6 myotubes (data not shown); (iii) induction of SNAT2 mRNA by AA deprivation occurs rapidly and is insensitive to the protein synthesis inhibitor cycloheximide, whereas induction of AS mRNA is inhibited by cycloheximide and is subject to a time delay following AA deprivation (consistent with ATF4 translation being a prerequisite for AS induction) (32) and (iv) synthetic AA (Me-AIB) and non-proteinic AA (sarcosine) are capable of inhibiting SNAT2 induction, although they are unlikely to prevent the accumulation of uncharged tRNAs for the proteinic AAs (and hence inhibit GCN2 activation).

The SNAT2 Sensor—A variety of AAs were incapable (Me-AIB/sarcosine) or poorly capable (Ala) of controlling MAPK signaling, although they potently repressed System A expression. All of these AAs interact with the System A carrier and as such we propose that SNAT2 is an AA sensor. Two compounds previously reported to interact with System A without repressing transporter expression (2,4-diaminobutyrate (33) and S-methyl-(L)-cysteine (19)) both inhibit and repress System A in L6 cells in our hands. The ability of SNAT2 to control gene expression was demonstrated using a combined shRNA and transport kinetics approach. The observation that the repressive effect of SNAT2 substrates correlates with their transport Km, combined with the discovery that anti-SNAT2 shRNA elevated the expression of a SNAT2 reporter gene, leads us to propose that SNAT2 represses its own expression through a signal at least partly responsive to the occupancy of the substrate binding site on SNAT2. Although we cannot totally exclude the possibility that a non-metabolizable SNAT2 substrate such as MeAIB has intracellular actions, the idea that AA sensing by SNAT2 occurs at the cell membrane is supported by several observations (noting that a change in external pH affects Na+-binding and substrate translocation by SNAT2, not the Km for AA binding (34): (i) extracellular acidification reduces both SNAT2 transport activity and intracellular amino acid concentrations in L6 cells (35), but does not invoke a SNAT2 adaptive regulatory response or impair the response to amino acid deprivation (see supplemental Fig. S5); (ii) external alkalinization increases SNAT2 transport activity but does not repress basal SNAT2 expression; (iii) extracellular MeAIB reduces intracellular AA concentrations to a greater extent than acidification (35), yet represses the SNAT2 adaptive response.

Kielland-Brandt and co-workers (13) have proposed a mechanistic model for transporter-like sensors responding to changes in nutrient availability, possibly applicable to SNAT2, which distinguishes between signaling and non-signaling conformations based on the orientation of the substrate binding site within the membrane. Signaling downstream of the SNAT2 sensor appears to be a dominant factor for regulation of SNAT2 gene transcription in skeletal muscle cells, because the JNK pathway remains activated when MeAIB/sarcosine exert their repressive actions. Wortmannin and LY-294002 inhibit adaptive regulation without inhibiting JNK (data not shown), so PI3K (or related kinases) may function in the signaling pathway downstream of the SNAT2 sensor.


Figure 7
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  		FIGURE 7.
Multi-sensor model for the regulation of System A in nutrient-deprived cells. AA deprivation activates distinct signaling pathways that stimulate transcription of the SLC38A2 gene and ultimately lead to the expression of SNAT2: dark gray boxes indicate pathways identified in non-muscle cells (see the text for a full description). The tripartite AA responsive domain in the first intron of SLC38A2 is as described in Ref. 7. SNAT2 (Sensor A) acts as a repressor of gene expression and regulates the (as yet) uncharacterized Pathway A. The SNAT2 transport cycle illustrated is based on our current model for this process: AA binding either precedes Na+ binding or substrate binding is unordered (34). The upper dashed arrow represents the return of the unloaded SNAT2 carrier to an exofacial orientation, the step proposed to be impaired by trans-inhibitory AAs (19). MAP kinase activation during AA deprivation (Pathway B) is modulated by a distinct sensor (Sensor B) responsive to large neutral AAs (except the branched chain AAs). Depending on cell type, either JNK or ERK may function through pathway B (8, 9). Activation of GCN2 signaling (Pathway C) is an important component of the response to AA deprivation in certain cell types (20, 29), but it appears neither necessary nor sufficient for adaptive regulation of System A in muscle cells. These pathways may also contribute to the destabilization of SNAT2 protein during AA sufficiency.

 
AA transporters exhibiting a dual transport/receptor function have been demonstrated in yeast and Drosophila (12, 36, 37). In yeast, in the presence of AAs, Ssy1p stimulates a proteolysis-dependent signaling pathway, which increases the expression of a variety of AA permeases (37). Similarly, in Drosophila PATH is a high affinity AA sensor which controls TOR signaling (36). The PATH sensor is part of a gene family (SLC36), which is very closely related to the System A/N gene family (SLC38), and it is therefore conceivable that SNAT2 is a mammalian orthologue of PATH. However, if this were the case we would expect SNAT2 substrates to stimulate mTOR, and neither Me-AIB (Fig. 4A) nor sarcosine (data not shown) had this effect at the concentration studied, although they both interacted with, and regulated the System A transporter. Conversely, given that a PAT-family transporter has signaling capacity in Drosophila, the observed effects of System A substrates on SNAT2 expression might be mediated through a PAT transporter rather than through SNAT2, given that the mammalian SLC36 transporters PAT1 and PAT2 share many substrates with SNAT2 (for example alanine and proline) (38). This also seems unlikely, given that in separate studies we find that: (i) PAT-type transport activity in L6 cells, identified as low flux β-alanine transport inhibited by various PAT substrates (L-Ala, D-Ala, β-Ala, AIB > Me-AIB), does not interact with certain SNAT2 substrates (notably sarcosine) known to contribute to System A adaptation; (ii) D-Ala and β-Ala, PAT-substrates which do not interact appreciably with SNAT2, have only a modest effect on adaptive regulation.

An important distinction between SNAT2 and Ssy1p/PATH is that the System A transporter contributes significantly to transmembrane AA flux, whereas Ssy1 and PATH can be considered either slow or non-transporting transporter homologues. A more appropriate comparison may perhaps be made with Gap1p, a yeast AA permease, which regulates intracellular signaling (12, 39) and also makes an appreciable contribution to membrane transport, particularly when faced with low extracellular AA levels. In the presence of AAs, Gap1p stimulates the cAMP/PKA signaling pathway and elicits changes in metabolism and expression of stress-responsive genes (12).

Several Sensor Pathways and Multiple Levels of Control—Our demonstration that SNAT2 is regulated at the level of protein stability adds another dimension to the control of a protein already known to be influenced at the levels of transcription, subcellular localization and at several steps of the transport cycle (reviewed in Ref. 1; and see Ref. 19 for a discussion of trans-inhibition). The observation that cycloheximide treatment of L6 myotubes for 4 h results in a decrease of System A activity in AA-containing, but not AA-free medium is consistent with the suggestion that SNAT2 protein is stabilized in an AA-dependent manner (see supplemental Fig. S1). A domain within the N terminus of SNAT2 appears to be critical for this stabilization. Domains have been identified in the intracellular hydrophilic regions of Gap1p and Ssy1p that propagate ligand-induced signaling (12) and similar regions may be present in the N terminus of SNAT2. There is very recent evidence (40) that polyubiquitination of SNAT2 by the ubiquitin ligase Nedd4 -2, a process identifying proteins as targets for proteasomal breakdown, may contribute to the normal turnover of SNAT2 at the cell surface. It is conceivable that inhibition of this process contributes to the stabilization of SNAT2 during AA withdrawal, noting that high extracellular AA levels cause internalization of yeast Gap1p from the plasma membrane, allowing it to be targeted for vacuolar proteolysis (39).

Any model which attempts to explain the control of System A must place the nutrient-dependent responses in the context of cellular and organism-wide nutrient homeostasis. For example, an apparent inconsistency in the literature is that removing a single AA from standard medium elevates SNAT2 expression (7), whereas total AA deprivation stimulates SNAT2 expression in a manner that can be prevented by individual AAs. Although neither SNAT2 nor the putative JNK-regulating receptor are expected to be saturated in the presence of a physiological mix of AAs (based on transport kinetics and Fig. 4A), removing histidine from this mix (as an example) should not lead to an appreciable decrease in transporter or receptor occupancy. The stimulation of SNAT2 observed in histidine-deprived cells (29, 30) may result from GCN2 activation, whereas the stimulation of SNAT2 expression observed in the absence of AA may be largely due to signaling downstream of SNAT2 or MAPK signaling. We anticipate a degree of synergy to occur between these pathways (because pathway-specific AA such as Me-AIB and Tyr each reduced luciferase activity to a level comparable with the AA-supplemented control cells in Fig. 1D) and as such, small changes in AA availability may have large effects on SNAT2 expression.

Model for the Amino Acid-dependent Control of System A—We have integrated our data with other recent studies to generate a general model for the regulation of System A by nutrient availability which includes repressive signaling pathways downstream of at least two AA sensing mechanisms, one of these being the SNAT2 transporter itself (see Fig. 7). In addition to its role as an amino acid sensor, SNAT2 may also provide a passive input to other nutrient signaling pathways (e.g. the mTOR and GCN2 pathways, which respond to intracellular AA or AA-tRNA levels, respectively), as a consequence of its transport function. The global role of SNAT2 in transmembrane AA flux should not be underestimated, given that it is more widely expressed than any of its close relatives (4), and, unlike the other widely expressed neutral AA transporters (Systems ASC and L) which function as equilibrative AA exchangers, it is a concentrative transporter (as a consequence of its unidirectional/Na+-coupled transport cycle). Indeed, it has been postulated that SNAT2 could drive the accumulation of non-System A substrates, through providing intracellular exchange substrates that could be traded for extracellular AA, such as leucine, by System L (tertiary-active transport; (41)). Our model allows for both extracellular (via SNAT2 and JNK) and intracellular (via GCN2) AA sensing to contribute to regulation of the cellular expression of SNAT2, which should allow transport activity to be regulated according to both cellular AA requirement and systemic nutrient levels.

SNAT2 mRNA is highly expressed in rat skeletal muscle (4) and in vitro assays have shown that muscle System A is activated by prior exercise, insulin and AA deprivation (42). Additionally, positron emission tomography has verified System A transport in human muscle (43). However, despite the above, the precise physiological role of muscle System A remains uncertain because rat hindlimb perfusion experiments revealed a minimal contribution of System A to alanine transport (44). This may be due to trans-inhibition of SNAT2 by the high intra-muscular glutamine concentration. An alternative possibility, highlighted by the current study, is that a key function of SNAT2 in skeletal muscle may be as a nutrient receptor as opposed to a transporter.


   FOOTNOTES
 
* This work was funded by the UK Biotechnology & Biological Sciences Research Council. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Table S1. Back

1 To whom correspondence should be addressed. Tel.: 44-1382-384969; Fax: 44-1382-385507; E-mail: h.s.hundal{at}dundee.ac.uk.

2 The abbreviations used are: AA, amino acid; AS, asparagine synthetase; CaR, extracellular Ca2+ receptor; CMV, cytomegalovirus (promoter); ERK, extracellular signal-regulated kinase; IRES, internal ribosome entry segment; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; Me-AIB, {alpha}-methylaminoisobutyric acid; PI3K, phosphinositide-3-kinase; SNAT2, sodium-dependent neutral amino acid transporter 2; S6K, ribosomal protein S6 kinase; UTR, untranslated region; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Jeff Erickson (LSU Health Sciences Centre, New Orleans, LA) for the rat SNAT2 cDNA and Vadivel Ganapathy (Medical College of Georgia, Augusta, GA) for the rat SNAT5 cDNA.



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 DISCUSSION
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