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Originally published In Press as doi:10.1074/jbc.M504983200 on December 20, 2005

J. Biol. Chem., Vol. 281, Issue 8, 4876-4886, February 24, 2006
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A Carboxyl-terminal Determinant of the Neuronal Glutamate Transporter, EAAC1, Is Required for Platelet-derived Growth Factor-dependent Trafficking*

Amanda L. Sheldon{ddagger}§1, Marco I. González{ddagger}2, and Michael B. Robinson{ddagger}3

From the Departments of {ddagger}Pediatrics, Pharmacology, and §Neuroscience, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, May 5, 2005 , and in revised form, December 16, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1), undergoes rapid regulation after treatment with platelet-derived growth factor (PDGF) or phorbol ester in C6 glioma cells and neurons. A large intracellular pool of EAAC1 exists, from which transporters are redistributed to the cell surface in response to these signals. Here we show that PDGF had no effect on subcellular localization of the glial glutamate transporter, GLT-1, after transfection into C6 glioma cells. Chimeras consisting of domains from EAAC1 or GLT-1 were used to investigate structural motifs involved in PDGF-dependent redistribution of EAAC1. PDGF did not induce trafficking of an EAAC1 chimera containing the carboxyl-terminal domain of GLT-1; however, it did induce trafficking of a GLT-1 chimera containing the carboxyl-terminal domain of EAAC1. A truncated mutant of EAAC1 lacking 10 carboxyl-terminal amino acids was responsive to PDGF, whereas a mutant lacking 20 residues was not. Alanine substitution mutagenesis in this region revealed a short motif, 502YVN504, necessary for regulated trafficking. This motif was also involved in protein kinase C-dependent trafficking, as mutant transporters exhibited an attenuated response to phorbol ester. Interestingly, the presence of YVN in the homologous region of a nonresponsive chimera was not sufficient to confer regulated trafficking; however, the presence of a 12-amino acid motif starting at this Tyr residue was sufficient to confer responsiveness to PDGF. These studies identify a novel motif within the carboxyl terminus of EAAC1 which is required for regulated trafficking. The possibility that this motif targets EAAC1 to an intracellular, "regulated pool" is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excitatory neurotransmission in the mammalian central nervous system is mediated predominantly by the acidic amino acid glutamate. In addition to mediating swift synaptic depolarization, glutamatergic neurotransmission is important for physiological processes such as synapse development and synaptic plasticity (1–3). However, high levels of extracellular glutamate result in excessive activation of glutamate receptors, a factor that presumably contributes to the cytotoxicity associated with stroke, hypoglycemia, and brain injury (4–6). Synaptic glutamate concentrations are tightly regulated by a family of high affinity, Na+-dependent glutamate transporters, ensuring crisp synaptic neurotransmission. This family consists of five members: GLAST4 (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (for review, see Refs. 7 and 8). In contrast to other neurotransmitter transporters, EAAC1 is expressed predominantly on postsynaptic neurons, where it is localized to perisynaptic membrane (9, 10). Here, uptake via EAAC1 is believed to prevent glutamate spillover and activation of extrasynaptic receptors (11). Antisense knock-down of the neuronal glutamate transporter revealed that transport by EAAC1 accounts for ~40% of glutamate uptake in the hippocampus (12), an area that is both extremely plastic and susceptible to excitotoxic insults. EAAC1 expression has also been found on presynaptic GABA terminals (9, 13), where the transporter is thought to supply glutamate as a precursor for GABA synthesis (14, 15). EAAC1 is also unique in that only ~30% of the transporter is expressed at the cell surface in both C6 glioma cells and primary neuronal cultures (16, 17). Protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA) or PDGF receptor activation results in an increase in EAAC1 at the cell surface in C6 glioma (16, 18) or primary neuronal cultures (17, 19). PDGF treatment results in approximately a 40–50% increase in EAAC1 at the cell surface, apparently from increased insertion of EAAC1 at the plasma membrane rather than decreased endocytosis. PMA has a larger effect on cell surface expression and appears both to increase insertion and to decrease endocytosis of EAAC1 (17).

The effect of PDGF on other glutamate transporters has not been examined. The bulk of glutamate uptake in the cortex is mediated by GLT-1, which is enriched on astrocytic processes that ensheath the synapse (for review, see Refs. 8 and 20). The mechanisms underlying PDGF-stimulated trafficking of EAAC1 as well as how signals are transduced by transporters to result in trafficking are unknown. In the present study, we sought to identify amino acid motifs in EAAC1 which are involved in PDGF-induced trafficking of EAAC1 to the cell surface. By identifying residues involved in this regulation, we hoped to elucidate mechanisms underlying PDGF-stimulated trafficking of EAAC1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—C6 glioma cells were obtained from the American Type Culture Collection (Rockville, MD). 10-cm cell culture plates were from Corning (Cambridge, MA). Dulbecco's modified Eagle's medium, trypsin-EDTA, L-glutamine, and penicillin-streptomycin were purchased from Invitrogen. Fetal bovine serum was obtained from Hyclone (Logan, UT). GenePorter transfection reagent was purchased from Gene Therapy Systems (San Diego, CA). Bovine serum albumin and PMA were purchased from Sigma. PDGF was purchased from Calbiochem. N-hydroxysulfosuccinimidobiotin (NHS-biotin), UltraLink immobilized monomeric avidin, and bicinchoninic acid (BCA) protein assay kit were purchased from Pierce. Protein G-agarose beads were from Invitrogen.

Rabbit polyclonal anti-actin and anti-Myc epitope antibodies were purchased from Sigma. Mouse monoclonal anti-Myc epitope antibody was purchased from Clontech (Palo Alto, CA). Mouse monoclonal anti-hemagglutinin (HA) antibody (clone 12CA5) was obtained from Roche Applied Science. Rabbit polyclonal anti-phosphotyrosine, mouse monoclonal anti-phosphotyrosine, and rabbit polyclonal anti-PDGF receptor beta antibodies were purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). Rabbit polyclonal anti-EAAC1 and GLT-1 antibodies were the generous gift of Dr. Jeffrey D. Rothstein (9). Rabbit polyclonal anti-interleukin-2 receptor (Tac) antibody was purchased from Santa Cruz Technology (Santa Cruz, CA). Anti-rabbit and anti-mouse horseradish peroxidase IgG, rainbow molecular weight marker, and enhanced chemiluminescence kits (ECL) were obtained from Amersham Biosciences. Immobilon P (polyvinylidene fluoride membrane) was purchased from Millipore (Bedford, MA).

Cell Culture—C6 glioma were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37 °C. Cells were used up to passage 60, and no gross morphological changes or changes in experimental effects were observed resulting from passage number.

Generation of Mutant Transporters—Chimeric glutamate transporters were constructed previously using cDNAs encoding GLT-1 and EAAC1 rat transporter subtypes (21). Myc-EAAC1 plasmid was constructed by subcloning full-length rat EAAC1 (amino acids 1–523) into the pCMV-Myc plasmid from Clontech, using XhoI and EcoRI restriction sites. Myc-EAAC1 truncated mutants were created by PCR amplification and subcloning into the pCMV-Myc plasmid, with Asp513 as the last amino acid of Myc-EAAC1{Delta}10 (amino acids 1–513) and Val503 as the last amino acid of Myc-EAAC1{Delta}20 (amino acids 1–503). Amino acid substitution mutants were created using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) and pCMV-Myc-EAAC1 or EEEG chimera templates. All sequences were verified by sequencing at the molecular biology core at the Children's Hospital of Philadelphia.

Transient Transfection of C6 Glioma Cells—C6 cells were grown to 50–60% confluence in 10-cm plates before the introduction of cDNA plasmids with a cationic lipid-based procedure, GenePorter. Cells were transfected with 6–10 µg of cDNA mixed with 30–50 µl of GenePorter in a 1 µg:5 µl ratio, ~18 h prior to experimental use.

Biotinylation of Cell Surface Transporters—C6 cells were serum-starved in Dulbecco's modified Eagle's medium containing 0.5% bovine serum albumin for 2 h prior to treatment with vehicle, PDGF (20 ng/ml), or PMA (100 nM). Cell surface proteins were then biotinylated as described previously (17, 18). Briefly, cell monolayers were rinsed twice with ice-cold PBS, pH 7.35, containing 0.1 mM CaCl2 and 1.0 mM MgCl2. The cells were then incubated in 2 ml of biotinylation solution (1 mg/ml NHS-biotin in PBS Ca/Mg) for 25 min at 4 °C with gentle agitation. The solution was aspirated, and excess biotin was quenched by incubating the cells with PBS Ca/Mg containing 100 mM glycine for 25 min at 4 °C with gentle shaking. Cells were then lysed in 1 ml of radioimmunoprecipitation assay buffer containing protease inhibitors (1 µg/ml leupeptin, 250 µM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, and 1 mM iodoacetamide) and phosphatase inhibitors (10 mM sodium fluoride, 30 mM sodium pyrophosphate, and 1 mM sodium orthovanadate). After removal of cellular debris by centrifugation, biotinylated proteins were batch extracted using UltraLink immobilized monomeric avidin (avidin beads). SDS-PAGE loading buffer was added to cell lysate, biotinylated proteins (cell surface proteins), and nonbiotinylated proteins (intracellular proteins). Each of these fractions was diluted and loaded such that the sum of immunoreactivity in the nonbiotinylated and biotinylated fractions should equal the immunoreactivity found in the lysate, if the yield from avidin extraction is 100%.

Measurement of Na+-dependent Transport Activity—Transport activity in C6 glioma was measured in 12-well plates as described previously (22). After preincubation with PDGF, C6 cells were assayed in a 37 °C water bath. Wells were rinsed twice with either 1 ml of warm Na+- or choline-containing buffer and then incubated with 0.5 µM L-[3H]glutamate for 5 min. These washes and incubation period result in a 10-min delay between the end of treatment with PDGF and conclusion of the measure of transporter activity. After stopping uptake of radioactive glutamate, the cells were solubilized, and samples were taken for analysis of radioactivity in a scintillation counter. Na+-dependent uptake was calculated as the difference in radioactivity accumulated in the presence and absence of Na+.

Immunoprecipitation—After treatment, C6 cells were washed twice with PBS Ca/Mg and then lysed in 1 ml of radioimmunoprecipitation buffer containing protease and phosphatase inhibitors (listed above). Lysates were precleared by shaking with 40 µl of protein-G agarose beads at 4 °C for 1 h, and then 450 µg of cell lysate protein was mixed overnight at 4 °C with 4 µg immunoprecipitation antibody (mouse monoclonal anti-Myc, rabbit polyclonal anti-EAAC1, mouse monoclonal anti-phosphotyrosine, or rabbit polyclonal anti-phosphotyrosine) or 4 µg of rabbit or mouse IgG. Proteins were precipitated by incubating with 25 µl of protein-G agarose beads for 2 h at 4 °C. Immunocomplexes were washed four times with radioimmunoprecipitation buffer and solubilized at 95 °C for 5 min in 25 µl of SDS sample buffer.

Western Blot Analysis—Proteins were resolved on 8% SDS-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and blocked for 1 h in TBS-T (50 mM Tris, pH 8.0, 150 mM NaCl, 0.2% Tween 20) containing 1% nonfat dry milk. Membranes were then probed with the appropriate primary antibody: anti-EAAC1 (1:75), anti-GLT-1 (1:10,000), anti-actin (1:5,000), anti-HA (1:500), monoclonal anti-Myc (1:500), polyclonal anti-Myc (1:1,000), or anti-PDGF receptor (1:1,000). Membranes were washed and then incubated with anti-rabbit or anti-mouse horseradish peroxidase IgG (1:5,000). Protein bands were visualized with ECL.

Immunoreactivity was quantified using NIH Image software. We routinely see an immunoreactive band at ~66 kDa, which corresponds to EAAC1 monomer, and a band at ~218 kDa, which corresponds to EAAC1 multimer (16, 23). Within each fraction, the optical densities of both monomer and multimer bands were summed and used to quantitate EAAC1 immunoreactivity. Quantitation of the monomer band alone yielded similar results. Transporter immunoreactivity in each fraction (lysate, cell surface, or intracellular fraction) was normalized to the amount of actin in the lysate fraction. The amount of transporter observed in a fraction was expressed as a percentage of transporter in the corresponding control (vehicle) fraction. Actin immunoreactivity was used both as a loading control and to ensure that cells were intact during the biotinylation procedure. Experiments in which biotinylated actin was greater than 30% (consistent with cell lysis) were excluded. Based on this criterion 7 of 163 experiments were excluded. In four experiments the control construct, Myc-EAAC1, did not respond to PDGF; therefore results for all constructs tested in those experiments were not included. Data are presented as the means ± S.E. and compared using one-way ANOVA with Bonferroni post hoc analysis or paired t test. These tests were employed to compare the effect of PDGF on chimeric/mutant transporters to the effect of PDGF on wild-type transporters. In addition, for each construct we compared the effects of PDGF (as a percentage) with the effects of vehicle (100%) using a one-sample t test.


Figure 1
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  		FIGURE 1.
Time course for effects of PDGF on EAAC1. C6 glioma were treated with vehicle (15 min) or PDGF (20 ng/ml) for varying times at 37 °C. A, after treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. A representative Western blot probed with EAAC1 and actin antibodies, shows the effects of PDGF on cell surface expression of EAAC1. B, summary of results of three independent experiments (mean ± S.E.). Immunoreactivity observed with PDGF treatment is expressed as a percentage of that observed with vehicle treatment (control). The percentage of biotinylated actin in untreated controls was 3 ± 1% and did not change with treatment. C, Na+-dependent L-[3H]glutamate transport was measured as described under "Experimental Procedures." A summary of results of three independent experiments performed in triplicate (mean ± S.E.) is shown. Data are expressed as a percentage of activity observed in cells treated with vehicle for 15 min.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Time Course of Effects of PDGF on EAAC1 Activity and Cell Surface Expression—Previously, we have shown that treating C6 glioma with PDGF results in an increase in Na+-dependent transport activity. This is associated with an increase in EAAC1 at the cell surface, as assessed by biotinylation and Western analysis (16). The central nervous system-derived C6 glioma cell line is an attractive system to study EAAC1 in isolation because it expresses EAAC1 but none of the other glutamate transporter subtypes (18). In past experiments, L-[3H]glutamate transport and EAAC1 cell surface immunoreactivity were measured after a 30-min treatment with PDGF; however, other time points were not tested. To determine when the effect of PDGF on EAAC1 was maximal, C6 cells were treated with PDGF for different times, and cell surface expression was measured by biotinylation and Western analysis. Treatment with vehicle alone had no effect on cell surface expression of EAAC1 at any time point (data not shown). PDGF increased cell surface expression of EAAC1 maximally (178 ± 17% of control) at 15 min (Fig. 1, A and B). PDGF increased glutamate transport activity maximally (183 ± 2% of control) at 5 min (Fig. 1C); however, multiple washes and a 5-min incubation period with radioactive glutamate resulted in a 10-min delay between the end of treatment with PDGF and conclusion of the measure of transporter activity. Therefore, the maximal increases occur at similar times, and subsequent biotinylation experiments were conducted after 15 min of treatment.


Figure 2
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  		FIGURE 2.
Effects of PDGF on EAAC1 and GLT-1 cell surface expression. C6 glioma were treated with vehicle or PDGF (20 ng/ml) for 15 min. After treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. A,C6 cells were transiently transfected with GLT-1 cDNA 18 h before PDGF treatment. A representative Western blot probed with GLT-1 and actin antibodies is shown. All lanes are from different parts of the same gel. Lysate from untransfected cells is included in the left lane. B, representative Western blot probed with EAAC1 and actin antibodies, showing the effect of PDGF on cell surface expression of EAAC1 in untransfected cells. C, summary of results of a minimum of four independent experiments (mean ± S.E.). ** p < 0.01 compared with percent change in cell surface expression of EAAC1 caused by PDGF (one-way ANOVA with Bonferroni post hoc analysis). ### p < 0.001 compared with cell surface expression of EAAC1 in vehicle-treated cells (one-sample t test). PDGF had no significant effect on GLT-1 cell surface expression (one-sample t test). For GLT-1 experiments, the percentage of biotinylated actin in untreated controls was 16 ± 3% and did not change with treatment. For EAAC1 experiments, the percentage of biotinylated actin in untreated controls was 7 ± 5% and did not change with treatment.

 
PDGF Stimulation Has a Differential Effect on EAAC1 and GLT-1— Although the effects of PDGF on EAAC1 are well documented, it was unknown whether PDGF affects the astroglial glutamate transporter, GLT-1. To determine the effect of PDGF on GLT-1, C6 cells were transiently transfected with a cDNA clone encoding GLT-1, and cell surface expression was assessed by biotinylation and Western analysis. Approximately 80% of GLT-1 was localized to the plasma membrane in the basal state (Table 1), and there was no increase in cell surface GLT-1 immunoreactivity with PDGF treatment (99 ± 6% of control) (Fig. 2, A and C). In additional experiments, 30% of endogenous EAAC1 was located at the cell surface in the basal state (Table 1), and PDGF increased cell surface expression of EAAC1 to 148 ± 4% of control (Fig. 2, B and C), in agreement with past studies (16, 17). Therefore, in contrast to its effects on EAAC1, PDGF had no effect on subcellular localization of GLT-1.


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  		TABLE 1
Basal biotinylated transporter and effects of PDGF on biotinylated transporter C6 glioma were transiently transfected with different transporter cDNAs. Basal biotinylated transporter is the amount of transporter in the biotinylated fraction after vehicle treatment, expressed as a percentage of total transporter. The effect of PDGF on cell surface expression is expressed as a percentage of that observed in vehicle-treated cells. Data are the mean ± S.E.; the number of independent observations is listed in parentheses.

 
The Carboxyl Terminus of EAAC1 Is Necessary for PDGF-induced Redistribution—Based on the recent elucidation of the crystal structure of a prokaryotic glutamate transporter homolog (24), these glutamate transporters are thought to contain eight membrane-spanning domains, with cytoplasmic amino and carboxyl termini. The mammalian members of this family share 50–60% sequence similarity (for review, see Ref. 8), suggesting that the differential effect of PDGF could be related to differences in primary structure of these transporters. Therefore, the differential effect of PDGF was used to define motifs of EAAC1 involved in PDGF-induced redistribution. In an earlier study, chimeras consisting of domains originating from EAAC1 or GLT-1 (Fig. 3A) were used to identify structural motifs required for PMA-induced regulation of GLT-1 (21). Chimeras containing the carboxyl terminus of EAAC1 were tagged with an HA epitope to distinguish them from endogenously expressed EAAC1. Because PDGF had no effect on cell surface expression of GLT-1, substitution of an EAAC1 domain with an equivalent domain of GLT-1 allowed us to determine whether that EAAC1 domain was necessary for PDGF-dependent translocation. C6 glioma were transfected with cDNAs encoding chimeric transporters, and trafficking was assessed by biotinylation of cell surface proteins and Western analysis. PDGF increased cell surface expression of HA-EAAC1 to 160 ± 12% of control (Fig. 3D), indicating that the HA tag did not interfere with this regulated trafficking of EAAC1. One problem we confronted is that C6 cells display a robust cross-reacting band to the HA antibody, which is directly above the transporter monomer band; however, we were able to resolve the HA-transporter monomer and multimer bands from this cross-reacting band. An untransfected lysate sample was always run in an adjacent lane to confirm the difference between the cross-reacting band and transporter bands. Similarly to GLT-1, all chimeras containing GLT-1 domains displayed a higher percentage of transporters at the cell surface in the basal state (Table 1). However, there was intracellular transporter available for redistribution. Initially, multiple chimeras were analyzed; for instance, HA-EEGE responded to PDGF similarly to EAAC1 (data not shown). However, the focus was soon switched to EEEG because of the apparent difference in response to PDGF compared with EAAC1. PDGF had no effect on cell surface expression of EEEG (98 ± 5% of control), which differed significantly from the percent change at the cell surface of endogenous EAAC1 or transfected HA-EAAC1 (Fig. 3, B and D). Therefore, residues within the 37 carboxyl-terminal amino acids of EAAC1 are required for PDGF-dependent redistribution. The reciprocal chimera, HA-GGGE, consists of three GLT-1 domains and the carboxyl terminus of EAAC1. PDGF increased cell surface expression of HA-GGGE to 139 ± 10% of control (Fig. 3, C and D), which was not significantly different from the PDGF-induced increase in cell surface EAAC1 or transfected HA-EAAC1. Therefore, the 37 carboxyl-terminal residues of EAAC1 are sufficient to confer PDGF-stimulated trafficking to another member of this transporter family.


Figure 3
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  		FIGURE 3.
Effects of PDGF on cell surface expression of chimeric transporters. A, schematic representation of chimeras consisting of domains originating from GLT-1 (denoted as G), shown in white boxes, or EAAC1 (denoted as E), shown in dark gray boxes. Numbers represent the number of amino acids in each domain. C6 cells were transiently transfected with chimeric transporter cDNAs; 18 h later cells were treated with PDGF (20 ng/ml) for 15 min. After treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. Lysates from untransfected cells are included in the left lane. B, representative Western blot for EEEG samples probed with GLT-1 and actin antibodies. C, representative Western blot for HA-GGGE samples probed with HA and actin antibodies. In this experiment most of the chimera migrated as a multimer. D, summary of results of a minimum of three independent experiments (mean ± S.E.). *** p < 0.001 compared with percent change in cell surface expression of EAAC1 caused by PDGF (one-way ANOVA with Bonferroni post hoc analysis). ### p < 0.001 compared with cell surface expression of transporter in vehicle-treated cells; ## p < 0.01 compared with cell surface expression of transporter in vehicle-treated cells (one-sample t test). PDGF had no significant effect on EEEG cell surface expression (one-sample t test). For HA-EAAC1 experiments, the percentage of biotinylated actin in untreated controls was 18 ± 3% and did not change with treatment. For EEEG experiments, the percentage of biotinylated actin in untreated controls was 22 ± 4% and did not change with treatment. For HA-GGGE experiments, the percentage of biotinylated actin in untreated controls was 13 ± 4% and did not change with treatment.

 
Amino Acids Asn504–Asp513 Are Required for PDGF-dependent Redistribution—Truncated mutants of EAAC1 were prepared to focus on the carboxyl terminus of EAAC1. These mutants were tagged with the Myc epitope because C6 glioma lysates did not display cross-reacting bands with the Myc antibody (Fig. 4A). C6 cells were transfected with Myc epitope-tagged EAAC1, lacking either the last 10 (Myc-EAAC1{Delta}10) or the last 20 (Myc-EAAC1{Delta}20) amino acids (Fig. 5A). PDGF-stimulated trafficking was assessed by biotinylation and Western analysis. PDGF increased Myc-EAAC1{Delta}10 at the cell surface to 155 ± 9% of control (Fig. 4, B and D); therefore, the last 10 amino acids of the carboxyl terminus (514–523) are not required for this redistribution. In contrast, PDGF did not increase Myc-EAAC1{Delta}20 cell surface expression (88 ± 8% of control; Fig. 4, C and D), demonstrating that amino acids Asn504–Asp513 are necessary for PDGF-stimulated trafficking. To determine whether this motif is specific for PDGF or more broadly required for regulated redistribution, the effects of PMA on truncated transporters were assayed. In past studies, PMA increased EAAC1 cell surface expression nearly 2-fold within 15 min (17). Likewise, we found that PMA increased cell surface expression of Myc-EAAC1{Delta}10 to 222 ± 15% of control, indicating that the last 10 residues are not required for the PMA-stimulated increase in cell surface expression (Fig. 4E). Interestingly, the PMA-induced increase in cell surface expression of Myc-EAAC1{Delta}20 was significantly attenuated (125 ± 11% of control) compared with the increase in Myc-EAAC1{Delta}10. However, one observation not easily explained is that PMA appeared to increase the amount of transporter in the lysate for all of the Myc-EAAC1 constructs. The increase was not specific to any mutant transporter (Figs. 4E and 5E) and was not observed in parallel experiments using PDGF treatment. It is unlikely that this effect resulted from new protein synthesis and maturation of the transporter because this would likely require more than 15 min. One possibility is that PMA increased the yield during preparation of the lysate. After biotinylation, cellular debris is cleared by centrifuging the lysate and removing the pellet. When the sample was sonicated before centrifugation the pellet was virtually eliminated, meaning transporter that may have been lost in the pellet before should now be incorporated in the sample. However, the increase in transporter in the lysate fraction with PMA treatment was still observed (data not shown). Another possibility is that PMA increases the immunoreactivity of the Myc epitope because it contains two serine residues that could theoretically be modified by PKC activation. To control for increased transporter immunoreactivity in the lysate, the cell surface fraction was also normalized to the amount of transporter in the lysate. When the cell surface fractions were normalized for Myc immunoreactivity in the lysate, PMA increased cell surface expression of Myc-EAAC1{Delta}10 to 157 ± 13% of control but did not increase Myc-EAAC1{Delta}20 cell surface expression (99 ± 13% of control), which was significantly different from Myc-EAAC1{Delta}10 (p < 0.01, one-way ANOVA with Bonferonni post hoc analysis). Therefore the results were similar regardless of the normalization method. Thus, it appears that amino acids Asn504–Asp513 are also required for PKC-stimulated redistribution.


Figure 4
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  		FIGURE 4.
Effects of PDGF or PMA on cell surface expression of Myc-tagged, truncated EAAC1. A, untransfected or Myc-EAAC1-transfected cell lysates were prepared and probed with Myc antibody. B, C6 cells were transfected with truncated Myc-EAAC1 cDNAs 18 h prior to PDGF (20 ng/ml) or PMA (100 nM) treatment for 15 min. After treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. A representative Western blot probed with Myc and actin antibodies shows the effects of PDGF or PMA on Myc-EAAC1{Delta}10 cell surface expression. C, representative Western blots probed with Myc and actin antibodies; notice that PDGF had no effect on Myc-EAAC1{Delta}20 cell surface expression. Intracellular and biotinylated fractions were run on the same gel, whereas lysate fractions were run on a second gel. D, summary of results of six independent experiments (mean ± S.E.). *** p < 0.001 compared with percent change in cell surface expression of Myc-EAAC1{Delta}10 caused by PDGF (one-way ANOVA with Bonferroni post hoc analysis). ## p < 0.01 compared with cell surface expression of Myc-EAAC1{Delta}10 in vehicle-treated cells (one-sample t test). E, summary of results of five independent experiments (mean ± S.E.). *** p < 0.001 compared with percent change in cell surface expression of Myc-EAAC1{Delta}10 caused by PMA (one-way ANOVA with Bonferroni post hoc analysis). ## p < 0.01 compared with cell surface expression of Myc-EAAC1{Delta}10 in vehicle-treated cells (one-sample t test). Neither PDGF nor PMA had a significant effect on Myc-EAAC1{Delta}20 cell surface expression compared with vehicle-treated cells (one-sample t test). For Myc-EAAC1{Delta}10, the percentage of biotinylated actin in untreated controls was 14 ± 5% and did not change with treatment. For Myc-EAAC1{Delta}20, the percentage of biotinylated actin in untreated controls was 15 ± 5% and did not change with treatment.

 
A Short Peptide Motif 502YVN504 Is Required for PDGF-stimulated Trafficking of EAAC1—To identify individual amino acids involved in PDGF-induced trafficking more specifically, this 10-amino acid stretch (Asn504–Asp513) was further analyzed using alanine screening mutagenesis. In addition, because a tyrosine residue is located two amino acids upstream of the end of Myc-EAAC1{Delta}20 (Fig. 5A), and there is the theoretical possibility that terminating the transporter at this point could disrupt normal function of these residues, these two residues, Tyr502 and Val503, were also included in this screen. Using alanine substitution mutagenesis, groups of three sequential amino acids were mutated to alanine in wild-type Myc-EAAC1. C6 glioma were transfected with Myc-EAAC1 or Myc-tagged AAA mutant transporters, and trafficking was assessed by biotinylation followed by Western analysis. The Myc tag did not interfere with stimulated trafficking, as PDGF increased cell surface expression of Myc-EAAC1 to 142 ± 10% of control (Fig. 5, B and D). The effects of PDGF on AAA mutant transporters were always measured in parallel with the effect of PDGF on wild-type Myc-EAAC1. PDGF increased cell surface expression of Myc-EAAC1(GGF-AAA) to 163 ± 18% of control (Fig. 5D), indicating that residues 505GGF507 are not required for this regulation. Although mutating either 508SVD510 or 511KSD513 to AAA appears to have slightly attenuated the PDGF-induced increase in transporter at the cell surface (Fig. 5D), neither was significantly different from Myc-EAAC1, and PDGF significantly increased surface expression compared with vehicle. Interestingly, when 502YVN504 was mutated to AAA, PDGF had no effect on cell surface expression of the transporter (98 ± 10% of control; Fig. 5, C and D), revealing amino acids 502YVN504 as critical for PDGF-stimulated trafficking. We also tested whether this motif is specific for PDGF, or whether these residues are involved in PKC-stimulated trafficking of EAAC1 as well. PMA only increased cell surface expression of Myc-EAAC1(YVN-AAA) to 161 ± 14% of control (Fig. 5E), which was significantly attenuated compared with the increase in Myc-EAAC1 in parallel experiments (216 ± 14% of control). Thus it appears these residues are also involved in PKC-induced trafficking of EAAC1. However, PMA increased Myc-EAAC1(YVN-AAA) at the cell surface above basal levels; therefore additional residues are probably involved in PKC-dependent regulation of EAAC1.


Figure 5
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  		FIGURE 5.
Effects of PDGF or PMA on Myc-tagged wild-type EAAC1 or AAA substitution mutants, and effects of PDGF on single Ala or Phe substitution mutants. C6 cells were transiently transfected with Myc-EAAC1 or Myc-tagged EAAC1 mutants. 18 h later cells were treated with PDGF (20 ng/ml) or PMA (100 nM) for 15 min. After treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. Blots were probed with Myc and actin antibodies. A, sequence of the last 25 carboxyl-terminal amino acids of EAAC1, with demarcations indicating where the sequence ends for Myc-EAAC1{Delta}10 and Myc-EAAC1{Delta}20. Amino acids shown in bold (Tyr502–Asp513) were mutated in groups of three to alanine. B, representative Western blot showing the effects of PDGF or PMA on biotinylated Myc-EAAC1. C, representative Western blot for Myc-EAAC1(YVN-AAA); notice that PDGF did not increase cell surface expression. D, summary of results of a minimum of seven independent experiments (mean ± S.E.). * p < 0.05 compared with percent change in cell surface expression of Myc-EAAC1 caused by PDGF (one-way ANOVA with Bonferronipost hoc analysis). ## p < 0.01 compared with cell surface expression of transporter in vehicle-treated cells; # p < 0.05 compared with cell surface expression of transporter in vehicle-treated cells (one-sample t test). E, summary of results of six independent experiments. * p < 0.05 compared with percent change in cell surface expression of Myc-EAAC1 caused by PMA in parallel experiments (paired t test). ### p < 0.001 compared with cell surface expression of transporter in vehicle-treated cells. ## p < 0.01 compared with cell surface expression of transporter in vehicle-treated cells. F, effects of PDGF on single Ala or Phe substitution mutants: summary of the results of a minimum of six independent experiments (mean ± S.E.). ## p < 0.01 compared with cell surface expression of transporter in vehicle-treated cells; # p < 0.05 compared with cell surface expression of transporter in vehicle-treated cells (one-sample t test). PDGF increased Myc-EAAC1(VN-AA), although it was not quite significant (p = 0.05, one-sample t test). The percentage of biotinylated actin in untreated controls is listed for each construct; it did not change with treatment. Myc-EAAC1, 12 ± 2%; Myc-EAAC1(YVN-AAA), 18 ± 3%; Myc-EAAC1(GGF-AAA), 16 ± 5%; Myc-EAAC1(SVD-AAA), 19 ± 3%; Myc-EAAC1(KSD-AAA), 20 ± 2%; Myc-EAAC1(Y502F), 2 ± 1%; Myc-EAAC1(V503A), 3 ± 2%; Myc-EAAC1(N504A), 5 ± 2%; Myc-EAAC1(VN-AA), 5 ± 3%.

 
EAAC1 Is Not Phosphorylated on Tyr Residues after PDGF Treatment— Next, the mechanism underlying the requirement of residues 502YVN504 for PDGF-stimulated trafficking of EAAC1 was explored. Phosphorylation of tyrosine within this motif could provide a functional link between PDGF receptor activation and transporter translocation. For instance, studies on the GABA transporter, GAT1, have provided strong evidence that phosphorylation of two tyrosine residues in GAT1 is required for the substrate-mediated increase in GAT1 at the cell surface, although in this case the increase results from decreased endocytosis (25). Possible PDGF-induced tyrosine phosphorylation of EAAC1 from untransfected or transfected C6 glioma was investigated using immunoprecipitation experiments. Initially, phosphorylation of endogenous EAAC1, Myc-EAAC1, or Myc-EAAC1(YVN-AAA) was measured after PDGF treatment by immunoprecipitating transporter and probing blots with anti-phosphotyrosine. Although reprobed blots indicate that EAAC1 (n = 1), Myc-EAAC1 (n = 2), or Myc-EAAC1(YVN-AAA) (n = 2) was successfully immunoprecipitated, there was no evidence of transporter phosphorylation on tyrosine residues, with or without PDGF treatment, for any of the EAAC1 variants tested (data not shown). Next, immunoprecipitation with monoclonal anti-phosphotyrosine from untransfected, Myc-EAAC1-transfected, or Myc-EAAC1(YVN-AAA)-transfected C6 cells was attempted, followed by immunoblotting with polyclonal anti-EAAC1 or anti-Myc antibody. Again, there were no immunoreactive transporter bands, with or without PDGF treatment, indicating that none of the transporters was pulled down with monoclonal anti-phosphotyrosine (n = 4 for EAAC1, n = 5 for Myc-EAAC1, n = 5 for Myc-EAAC1(YVN-AAA); data not shown). As a positive control, in each experiment blots were stripped and reprobed with anti-PDGF receptor beta antibody. In samples treated with PDGF, a robust immunoreactive band at 190 kDa corresponding to phosphorylated PDGF receptor beta was observed, providing strong evidence that the monoclonal anti-phosphotyrosine antibody was able to pull down phosphorylated protein. In parallel, immunoprecipitation experiments from transfected C6 cells were carried out with polyclonal anti-phosphotyrosine antibody, and blots were probed with monoclonal anti-Myc. A faint immunoreactive band corresponding to Myc-EAAC1 was present in samples in which rabbit IgG antibody was used for immunoprecipitation as a negative control; therefore, there was a low level of Myc-EAAC1 that was immunoprecipitated nonspecifically. Sometimes this band was present in samples from Myc-EAAC1 transfected cells, in which polyclonal anti-phosphotyrosine antibody was used as the immunoprecipitation antibody (n = 6, data not shown). However, this was not a consistent finding, and the signal was about the same intensity as that found in the IgG lane. In addition, this signal did not change with PDGF treatment and was not abolished in samples from Myc-EAAC1(YVN-AAA)-transfected cells. Therefore, polyclonal anti-phosphotyrosine was most likely immunoprecipitating a low level of transporter nonspecifically. Taken together, the results from the immunoprecipitation experiments strongly suggest that EAAC1 does not exhibit basal tyrosine phosphorylation, and PDGF treatment does not result in detectable tyrosine phosphorylation of EAAC1. In addition, these results argue against the hypothesis that phosphorylation of Tyr within 502YVN504 is a necessary step for EAAC1 trafficking.

Mutation of Single Amino Acids within 502YVN504 Does Not Abolish PDGF-dependent Regulation of EAAC1—Because it appeared that phosphorylation of Tyr502 within the 502YVN504 motif is not required for PDGF-dependent trafficking of EAAC1, we next sought to determine whether any residues within the motif were individually required for PDGF-induced regulation. Using alanine substitution mutagenesis, one double alanine mutant and three single mutants were prepared. Val503–Asn504 were mutated to Ala-Ala, Val503 was mutated to Ala, and Asn504 was mutated to Ala. Instead of replacing Tyr502 with Ala, the tyrosine residue was mutated to Phe because the phenylalanine structure more closely resembles that of tyrosine. C6 glioma were transfected with Myc-EAAC1 or Myc-tagged mutant transporters, and trafficking was assessed by biotinylation followed by Western analysis. The effects of PDGF on mutant transporters were again measured in parallel with the effect of PDGF on wild-type Myc-EAAC1. PDGF increased cell surface expression of Myc-EAAC1 to 125 ± 7% of control (Fig. 5F). Surprisingly, PDGF increased cell surface expression of each mutant transporter: Myc-EAAC1(Y502F), Myc-EAAC1(V503A), Myc-EAAC1(N504A), and Myc-EAAC1(VN-AA) (Fig. 5F). Therefore, it appears that disruption of the entire 502YVN504 motif is necessary to abolish PDGF-dependent trafficking of EAAC1.

Amino Acids Tyr502–Asp513 Are Sufficient to Confer PDGF-induced Trafficking to EEEG—Interestingly, when the sequences for EAAC1 and GLT-1 are aligned, 502YVN504 within EAAC1 aligns with 538YAA540 within GLT-1. Because the nonresponsive chimeric transporter, EEEG, contains the carboxyl terminus of GLT-1, and therefore YAA instead of YVN, we determined whether mutating AA to VN in the EEEG backbone could rescue the response to PDGF (Fig. 6A). The EEEG(2) construct was prepared using site-directed mutagenesis, and transporter trafficking was assessed by biotinylation and Western analysis. PDGF did not increase cell surface expression of EEEG(2) (95 ± 9% of control; Fig. 6C). Likewise, PDGF did not significantly increase cell surface expression of two mutants containing further substitutions of EAAC1 residues for GLT-1 residues in the EEEG backbone (EEEG(5) or EEEG(7)). However, when 10 amino acids were mutated in EEEG (EEEG(10)) so that the entire 12-amino acid motif of EAAC1 was now present in the carboxyl terminus of EEEG, PDGF significantly increased cell surface expression of the transporter (121 ± 8% of control; Fig. 6, B and C). Therefore, this motif identified in EAAC1, 502YVNGGFSVDKSD513, is sufficient to confer PDGF-stimulated trafficking to a nonresponsive transporter.


Figure 6
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  		FIGURE 6.
Effects of PDGF on EEEG substitution mutants. C6 cells were transiently transfected with EEEG(2), EEEG(5), EEEG(7), or EEEG(10). 18 h later cells were treated with PDGF (20 ng/ml) for 15 min. After treatment, cell surface proteins were biotinylated and batch extracted with avidin beads at 4 °C, as described under "Experimental Procedures." Cellular fractions were analyzed by Western blot. A, alignment of the residues within the carboxyl termini of GLT-1 and EAAC1. GLT-1 residues within the carboxyl terminus of EEEG were mutated to the corresponding residues within EAAC1. B, representative Western blot for EEEG(10) samples probed with GLT-1 and actin antibodies. C, summary of results of a minimum of four independent experiments (mean ± S.E.). # p < 0.05 compared with cell surface expression of transporter in vehicle-treated cells (one-sample t test). The percentage of biotinylated actin in untreated controls is listed for each construct; it did not change with treatment. EEEG(2), 5 ± 2%; EEEG(5), 4 ± 1%; EEEG(7), 2 ± 1%; EEEG(10), 0%.

 
Although these studies suggest that this 12-amino acid motif may be sufficient to confer regulated trafficking to a nonresponsive transporter, we cannot rule out the possibility that other conserved motifs are also required. Therefore, we introduced these 12 residues onto the carboxyl terminus of an unrelated membrane protein, interleukin-2 receptor (Tac). The sequences of both cDNAs were verified by sequencing. Neither wild-type Tac nor the mutant Tac protein was detected by Western blot after transfection in C6 cells using an anti-Tac antibody. The amounts of cDNA (6–10 µg) and transfection times (18–48 h) were varied, but no expression was observed. As a positive control, we verified that the Tac antibody recognized Tac after transfection in a cell line that tends to express robust amounts of protein (human embryonic kidney 293 cells). Therefore, at present it is not possible to determine whether this motif is sufficient to confer PDGF-dependent trafficking onto an unrelated protein using C6 glioma.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we determined that PDGF had different effects on EAAC1 and GLT-1; PDGF increased cell surface expression of EAAC1, whereas it had no effect on subcellular localization of GLT-1. GLT-1 is responsible for the bulk of glutamate uptake throughout the cortex (for review, see Ref. 8), whereas the relative contribution of EAAC1 to glutamate transport is higher in the hippocampus, where it may account for ~40% of glutamate uptake (12). Another difference is the availability of a large intracellular pool of EAAC1 in neurons (13, 18). EAAC1 may be uniquely positioned to play a more specialized role in glutamate uptake at excitatory synapses. For instance, Levenson et al. (2) found that induction of long-term potentiation was associated with translocation of EAAC1 from inside the cell to the cell surface and that contextual fear conditioning also increased glutamate uptake and EAAC1 expression at the plasma membrane. Interestingly, Beckmann et al. (26) reported in a recent abstract that adeno-associated viral-mediated antisense knock-down of EAAC1 in rat hippocampus abolished long-term potentiation following high frequency stimulation. The contrasting effect of PDGF on EAAC1 and GLT-1 suggests that these two transporters can be regulated differentially, providing a mechanism to shift clearance of glutamate through these two different cell types (neurons and glia).

The glucose transporter GLUT4 undergoes a similar type of regulation in response to insulin in adipocytes (for review, see Ref. 27). Various investigators have tried to identify GLUT4 domains important for this regulation. To date, there is no evidence of direct modification of residues within GLUT4 related to insulin-stimulated trafficking, whereas a number of studies have identified motifs important for targeting GLUT4 to a regulated pool. It appears that motifs within both the amino and carboxyl termini are important for GLUT4 trafficking, with the amino-terminal FQQI motif seemingly involved in sorting GLUT4 out of early endosomes (28, 29) and a dileucine motif possibly functioning with neighboring acidic residues to sort GLUT4 to the glucose storage compartment (29, 30). Additional motifs within the carboxyl terminus have also been identified as important for retention in the glucose storage compartment (31).

Interestingly, the carboxyl terminus of EAAC1 was found to be necessary for transporter trafficking to the cell surface in response to PDGF receptor activation, as PDGF did not increase cell surface expression of the chimera, EEEG. In contrast, PDGF did increase cell surface expression of the reciprocal chimera, HA-GGGE. Although substitution with the carboxyl terminus of EAAC1 appeared to confer PDGF-stimulated trafficking to a normally nonresponsive transporter, GLT-1, it is possible that other conserved regions within GLT-1 may also be required.

At first we thought that perhaps the basal subcellular localization affected the responsiveness of the transporter to PDGF. Most of GLT-1 is expressed at the cell surface, whereas ~70% of EAAC1 is available intracellularly. Like GLT-1, chimeric transporters also displayed a higher percentage of transporters at the cell surface. However, even though ~78% of HA-GGGE was expressed at the cell surface in the basal state, intracellular transporters could still be mobilized in response to PDGF receptor activation. This led us to hypothesize that amino acid motifs present within the carboxyl terminus of EAAC1 were involved in PDGF-stimulated trafficking of the transporter.

Within the carboxyl terminus, we found that amino acids Asn504–Asp513 were required for stimulated trafficking of EAAC1, as PDGF increased cell surface expression of Myc-EAAC1{Delta}10 but not Myc-EAAC1{Delta}20. These residues were also involved in PKC-induced trafficking because the effect of PMA on Myc-EAAC1{Delta}20 cell surface expression was significantly attenuated. We next identified a short amino acid motif, 502YVN504, that is essential for PDGF-stimulated trafficking, as PDGF treatment had no effect on subcellular localization of Myc-EAAC1(YVN-AAA). Although they were not significantly different from the PDGF-induced increase in Myc-EAAC1, Myc-EAAC1(SVD-AAA) and Myc-EAAC1(KSD-AAA) both exhibited a trend toward an attenuated response to PDGF, indicating that in addition to 502YVN504, other residues within the larger motif 502YVNGGFSVDKSD513 may also be involved in the PDGF-dependent regulation. In support of this, the entire motif, Tyr502–Asp513, was necessary to confer PDGF-induced trafficking to a nonresponsive transporter, EEEG. We next investigated whether the requirement for 502YVN504 was specific to PDGF-stimulated trafficking or whether the motif was more broadly involved in regulated trafficking. Interestingly, this motif also appears to be involved in PKC-induced trafficking because the PMA-stimulated increase in Myc-EAAC1(YVN-AAA) at the cell surface was significantly attenuated.

We next sought to determine whether individual residues within the 502YVN504 motif were required for PDGF-induced translocation of EAAC1. We found that mutating either Val503 or Asn504 individually to alanine, or mutating Val503–Asn504 to Ala–Ala did not disrupt PDGF-dependent trafficking. In addition, mutation of Tyr502 to phenylalanine also did not disrupt PDGF-dependent trafficking of EAAC1. Therefore the minimal motif whereupon mutation to alanine disrupts PDGF-dependent trafficking is 502YVN504. However, 502YVN504 is not the minimal motif sufficient to confer PDGF-dependent trafficking to the nonresponsive transporter, EEEG. All 12 amino acids of EAAC1, Tyr502–Asp513, are required in EEEG for the transporter to respond to PDGF.

There are two ways in which this motif may be involved in PDGF-stimulated trafficking of EAAC1. First, the residues may be directly modified by signaling molecules downstream of PDGF, leading to translocation of EAAC1. Second, this motif may be required for regulated trafficking by targeting EAAC1 to an intracellular, regulated pool. Recently, Fournier et al. (17) provided evidence that separate intracellular pools may support constitutive and regulated trafficking of EAAC1. In these experiments, constitutive recycling and regulated trafficking of EAAC1 displayed differential sensitivity to temperature. Lowering assay temperature to 18 °C selectively inhibited the PDGF- or PMA-induced increase in cell surface EAAC1 but had no effect on constitutive recycling. This suggests that two factors may be required for regulation, the first being loading or sorting EAAC1 to a regulated pool and the second being a way in which this pool can be mobilized in response to these signals. The effects of PDGF and PMA are not additive (16); this is consistent with the possibility that both signaling pathways converge on the same finite pool of transporter available for redistribution.

Direct modification of 502YVN504 by phosphorylation was tested in immunoprecipitation experiments. There was no clear evidence of tyrosine phosphorylation of EAAC1 either before or after PDGF treatment, weakening the hypothesis that PDGF stimulates trafficking by a mechanism that depends on direct tyrosine phosphorylation. The results with polyclonal anti-phosphotyrosine were somewhat ambiguous, but we concluded that a specific association of the transporter with the phosphotyrosine antibody could not be detected. Taken together with the negative results using the monoclonal anti-phosphotyrosine antibody, EAAC1 does not appear to be phosphorylated on tyrosine residues in the basal state or after PDGF treatment. Therefore, it is unlikely that phosphorylation of Tyr502 is required for PDGF-induced trafficking of EAAC1. In support of this, Tyr within 502YVN504 is conserved in GLT-1 (a transporter that does not respond to PDGF), whereas Val and Asn are not. Further, PDGF increased cell surface expression of Myc-EAAC1(Y502F), demonstrating that Tyr phosphorylation on Tyr502 must not be required for PDGF-dependent regulation.

Another possibility is that the 12-amino acid motif functions to target EAAC1 to an intracellular, regulated pool. Within this motif, residues 502YVN504 were critical for the response of EAAC1 to PDGF. Although the amount of transporter at the cell surface in the basal state was equivalent for EAAC1, Myc-EAAC1, or Myc-EAAC1(YVN-AAA), indicating that the same amount of transporter was available in the cytosol, biotinylation only serves to separate cell surface proteins from intracellular proteins. It cannot be employed to differentiate among pools within the intracellular transporter population. In support of the targeting hypothesis, the effects of either PDGF or PMA were disrupted in both Myc-EAAC1{Delta}20 and Myc-EAAC1(YVN-AAA), providing evidence that these residues may be involved in directing EAAC1 to an intracellular, regulated pool. The effect of PDGF on cell surface expression was completely abolished, whereas the effect of PMA on cell surface expression was only attenuated. However, this is not overly surprising; a recent study indicates that PMA and PDGF may affect the kinetics of regulated trafficking of EAAC1 differently. Both PDGF and PMA appear to increase the rate of delivery of EAAC1 to the plasma membrane; however, in addition to the effects on delivery, PMA appears to decrease endocytosis of the transporter (17). PMA stimulation results in a larger increase in cell surface expression of EAAC1 than PDGF does, which may possibly result from the dual effect of PMA on insertion and stabilization of EAAC1 at the plasma membrane. Also, different domains may be required for the effects of PMA on insertion versus plasma membrane stabilization of EAAC1. Thus, the effect of PMA on plasma membrane stabilization of the transporter may account for the partial effect of PMA on Myc-EAAC1(YVN-AAA). Therefore, it is possible that amino acids 502YVN504 are critical as part of a 12-amino acid motif functioning to target EAAC1 to an intracellular pool that can be regulated by either signaling molecule. Because mutation of the entire 502YVN504 motif is required to abolish PDGF-dependent trafficking, one possibility is that the motif acts as a binding site for protein-protein interactions that are required for targeting the transporter to a regulated pool.

Interestingly, the 12-amino acid motif is also present in a larger, previously identified motif, 501KSYVNGGFAVD511. Cheng et al. (32) demonstrated that this motif is a sorting motif required for apical polarization of the human homolog of EAAC1, EAAT3, in Madin-Darby canine kidney cells and dendritic targeting in hippocampal neurons. Within this sorting motif, they found Val504–Asn505 and Phe508–Ala509 to be critical residues for apical polarization of EAAT3. The sorting motif overlaps with the Tyr502–Asp513 motif identified in this paper as sufficient for PDGF-stimulated trafficking of a nonresponsive transporter. So, overlapping motifs within the cytoplasmic carboxyl-terminal tail appear important both for regulated trafficking of EAAC1 and constitutive sorting of EAAT3. In fact, these motifs may perform overlapping functions of apical localization and targeting to a regulated pool.

In summary, we found that the astroglial glutamate transporter, GLT-1, does not traffic to the cell surface in response to PDGF treatment. We also provided evidence that the cytoplasmic carboxyl terminus of EAAC1 is necessary for PDGF-dependent trafficking to the plasma membrane. In addition, we identified 502YVNGGFSVDKSD513 within the carboxyl terminus as necessary for regulated trafficking and sufficient to confer PDGF-stimulated trafficking to a nonresponsive transporter. Within this motif, residues 502YVN504 were critical for the regulation. We also provide strong evidence that phosphorylation of tyrosine within this motif is not required for PDGF-dependent redistribution of EAAC1. The involvement of this motif does not appear specific to PDGF-stimulated trafficking; rather, it may target the transporter to an intracellular pool, which can be regulated by activation of either PDGF receptor or PKC.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants NS29868 and NS39011. Sequencing of clones was partially supported by Institutional Mental Retardation and Developmental Disabilities Research Center Grant P30-HD29679. 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

1 Partially supported by National Institutes of Health Grants 5-T32-GM07517 and 1-F31-MH1071008-01. Back

2 Partially supported by American Heart Association Grant 0325614U. Back

3 To whom correspondence should be addressed: Dept. of Pediatrics, 502N Abramson Pediatric Research Bldg., 3615 Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-2205; Fax: 215-590-3779; E-mail: Robinson{at}pharm.med.upenn.edu.

4 The abbreviations used are: GLAST, glutamate/aspartate transporter; ANOVA, analysis of variance; CMV, cytomegalovirus; EAAC1, excitatory amino acid carrier 1; EAAT1, 2, 3, 4, and 5, excitatory amino acid transporter 1, 2, 3, 4, and 5, respectively; EEEG, chimera of three domains from EAAC1 and the final domain from GLT-1; GABA, {gamma}-aminobutyric acid; GAT1, {gamma}-aminobutyric acid transporter subtype 1; GGGE, chimera of three domains from GLT-1 and the final domain from EAAC1; GLT-1, glutamate transporter 1; HA, hemagglutinin; PDGF, platelet-derived growth factor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Avtandil Kalandadze for preparing the chimeric transporters and Dr. Jeffrey Rothstein for the generous gift of anti-EAAC1 and anti-GLT-1 antibodies. We also thank Elizabeth Krizman for performing the measurements of glutamate uptake. We thank Dr. Mary E. Putt for valuable advice regarding statistical analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Monaghan, D. T., Bridges, R. J., and Cotman, C. W. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 365-402[CrossRef][Medline] [Order article via Infotrieve]
  2. Levenson, J., Weeber, E., Selcher, J. C., Kategaya, L. S., Sweatt, J. D., and Eskin, A. (2001) Nat. Neurosci. 5, 155-161
  3. Tsvetkov, E., Shin, R. M., and Bolshakov, V. Y. (2004) Neuron 41, 139-151[CrossRef][Medline] [Order article via Infotrieve]
  4. Choi, D. W. (1992) J. Neurobiol. 23, 1261-1276[CrossRef][Medline] [Order article via Infotrieve]
  5. Palmer, A. M., Marion, D. W., Botscheller, M. L., Swedlow, P. E., Styren, S. D., and DeKosky, S. T. (1993) J. Neurochem. 61, 2015-2024[Medline] [Order article via Infotrieve]
  6. Nilsson, P., Laursen, H., Hillered, L., and Hansen, A. J. (1996) J. Cereb. Blood Flow Metab. 16, 262-270[CrossRef][Medline] [Order article via Infotrieve]
  7. Sims, K. D., and Robinson, M. B. (1999) Crit. Rev. Neurobiol. 13, 169-197[Medline] [Order article via Infotrieve]
  8. Danbolt, N. C. (2001) Prog. Neurobiol. 65, 1-105[CrossRef][Medline] [Order article via Infotrieve]
  9. Rothstein, J. D., Martin, L., Levey, A. I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N., and Kuncl, R. W. (1994) Neuron 13, 713-725[CrossRef][Medline] [Order article via Infotrieve]
  10. He, Y., Janssen, W. G. M., Rothstein, J. D., and Morrison, J. H. (2000) J. Comp. Neurol. 418, 255-269[CrossRef][Medline] [Order article via Infotrieve]
  11. Diamond, J. S. (2001) J. Neurosci. 21, 8328-8338[Abstract/Free Full Text]
  12. Rothstein, J. D., Dykes-Hoberg, M., Pardo, C. A., Bristol, L. A., Jin, L., Kuncl, R. W., Kanai, Y., Hediger, M., Wang, Y., Schielke, J. P., and Welty, D. F. (1996) Neuron 16, 675-686[CrossRef][Medline] [Order article via Infotrieve]
  13. Conti, F., DiBiasi, S., Minelli, A., Rothstein, J. D., and Melone, M. (1998) Cereb. Cortex 8, 108-116[Abstract/Free Full Text]
  14. Sepkuty, J. P., Cohen, A. S., Eccles, C., Rafiq, A., Behar, K., Ganel, R., Coulter, D. A., and Rothstein, J. D. (2002) J. Neurosci. 22, 6372-6379[Abstract/Free Full Text]
  15. Mathews, G. C., and Diamond, J. S. (2003) J. Neurosci. 23, 2040-2048[Abstract/Free Full Text]
  16. Sims, K. D., Straff, D. J., and Robinson, M. B. (2000) J. Biol. Chem. 275, 5228-5237[Abstract/Free Full Text]
  17. Fournier, K. M., González, M. I., and Robinson, M. B. (2004) J. Biol. Chem. 279, 34505-34513[Abstract/Free Full Text]
  18. Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485[Abstract/Free Full Text]
  19. González, M. I., Kazanietz, M. G., and Robinson, M. B. (2002) Mol. Pharmacol. 62, 901-910[Abstract/Free Full Text]
  20. Robinson, M. B. (1999) Neurochem. Int. 33, 479-491[CrossRef]
  21. Kalandadze, A., Wu, Y., and Robinson, M. B. (2002) J. Biol. Chem. 277, 45741-45750[Abstract/Free Full Text]
  22. Dowd, L. A., and Robinson, M. B. (1996) J. Neurochem. 67, 508-516[Medline] [Order article via Infotrieve]
  23. Haugeto, Ø., Ullensveng, K., Levy, L. M., Chaudhry, F. A., Honore, T., Neilsen, M., Lehre, K. P., and Danbolt, N. C. (1996) J. Biol. Chem. 271, 27715-27722[Abstract/Free Full Text]
  24. Yernool, D., Boudker, O., Jin, Y., and Gouaux, E. (2004) Nature 431, 811-818[CrossRef][Medline] [Order article via Infotrieve]
  25. Whitworth, T. L., and Quick, M. W. (2001) J. Biol. Chem. 276, 42932-42937[Abstract/Free Full Text]
  26. Beckmann, M. L., Leary, G., Awes, A. N., Esslinger, C., Poulsen, D., Babcock, M., Manzoni, O., and Kavanaugh, M. P. (2005) Neuropharmacol. Conf. 15, Abstr. O25
  27. Watson, R. T., Kanzaki, M., and Pessin, J. E. (2004) Endocr. Rev. 25, 177-204[Abstract/Free Full Text]
  28. Palacios, S., Lalioti, V., Martinez-Arca, S., Chattopadhyay, S., and Sandoval, I. V. (2001) J. Biol. Chem. 276, 3371-3383[Abstract/Free Full Text]
  29. Melvin, D. R., Marsh, B. J., Walmsley, A. R., James, D. E., and Gould, G. W. (1999) Biochemistry 38, 1456-1462[CrossRef][Medline] [Order article via Infotrieve]
  30. Martinez-Arca, S., Lalioti, V. S., and Sandoval, I. V. (2000) J. Cell Sci. 113, 1705-1715[Abstract]
  31. Shewan, A. M., Marsh, B. J., Melvin, D. R., Martin, S., Gould, G. W., and James, D. E. (2000) Biochem. J. 350, 99-107
  32. Cheng, C., Glover, G., Banker, G., and Amara, S. G. (2002) J. Neurosci. 22, 10643-10652[Abstract/Free Full Text]

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