Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3–18

Gene 292 (2002) 81–90 www.elsevier.com/locate/gene

Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3–18 Matthew E.R. Butchbach a,b, Liching Lai a, Chien-liang Glenn Lin a,b,c,d,* a

Department of Neuroscience, The Ohio State University, 4068 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210-1239, USA Ohio State Biochemistry Program, The Ohio State University, 085 Botany and Zoology Building, 1735 Neil Avenue, Columbus, OH 43210-1220, USA c Neuroscience Graduate Studies Program, The Ohio State University, 4068 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210-1239, USA d Integrated Biomedical Science Graduate Program, The Ohio State University, 1190 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210-1239, USA b

Received 21 November 2001; received in revised form 8 April 2002; accepted 30 April 2002 Received by S. Salzberg

Abstract Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na 1-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3interacting protein called GTRAP3–18 has been identified by a yeast two-hybrid screening. GTRAP3–18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3–18, we cloned the mouse orthologue to GTRAP3–18 and determined its gene structure and its expression pattern. GTRAP3–18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3–18 genes contain three exons separated by two introns. The GTRAP3–18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3–18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3–18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3–18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Glutamate uptake; Brain; Untranslated regions; Immunohistochemistry; Comparative genomics

1. Introduction Glutamate is a dicarboxylic amino acid that is important for polypeptide biosynthesis, nitrogen turnover, energy Abbreviations: bp, base pairs; CaMKII, calcium-calmodulin-dependent protein kinase II; cDNA, DNA complementary to RNA; cpm, counts per minute; DAB, 3,3 0 -diaminobenzidine; dpm, disintegrations per minute; EAAT, excitatory amino acid transporter; GABA, g-aminobutyric acid; GST, glutathione-S-transferase; GTRAP3–18, glutamate transport-associated protein for EAAT3; kDa, kilodaltons; mCi, microCurie; Mr, relative molecular mass; NGS, normal goat serum; nt, nucleotides; ORF, open reading frame; PKA, protein kinase A; PKC, protein kinase C; PVDF, polyvinylidene difluoride; 3 0 RACE, rapid amplification of cDNA 3 0 end; RLM-5 0 RACE, RNA ligase-mediated rapid amplification of cDNA 5 0 end; RT-PCR, reverse transcriptase-polymerase chain reaction; PA, polyacrylamide; pAb, polyclonal antibody; PBS, phosphate-buffer saline; SDS, sodium dodecylsulfate; TBS, Tris-buffered saline; UTR, untranslated region * Corresponding author. Tel.: 11-614-688-5433; fax: 11-614-688-8742. E-mail address: [email protected] (C.G. Lin).

metabolism, amino acid transport, antioxidant synthesis and neurotransmission. In fact, glutamate is the major excitatory neurotransmitter used in the mammalian nervous system. Na 1-dependent glutamate transporters are responsible for the uptake of glutamate into the cell. Five mammalian Na 1-dependent glutamate transporters have been cloned to date: excitatory amino acid transporter 1 (EAAT1) (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1), EAAT4 and EAAT5 (Danbolt, 2001). EAAT3 is present in many peripheral tissues including the kidney and heart (King et al., 2001; Shayakul et al., 1999). Within the nervous system, EAAT1 and EAAT2 are found primarily on astrocytes while EAAT3, EAAT4 and EAAT5 are found principally on neurons. EAAT3 has been localized to the presynaptic cleft and non-synaptic areas of glutamatergic and GABAergic neurons (Coco et al., 1997; Conti et al., 1998; Rothstein et al., 1994). Functional ablation of EAAT3 in the rat by antisense oligonucleotide administration results in epilepsy and some motor impairment (Rothstein et al., 1996). GABA

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00669-8

82

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

levels were significantly reduced in specific brain regions of rats chronically treated with an EAAT3 antisense oligonucleotide (Eccles et al., 1996). The EAAT3 knockout mice, however, do not develop spontaneous seizures but show reduced spontaneous locomotor activity (Peghini et al., 1997). Interestingly, these mice develop dicarboxylic aminoaciduria as characterized by a 1400-fold increase in urinary glutamate levels (Peghini et al., 1997). These studies suggest that the loss of functional EAAT3 can lead to neurological as well as renal abnormalities. The expression and function of glutamate transporters can be regulated by activation of intracellular signaling molecules. For example, the surface expression of EAAT3 can be increased by activation of protein kinase C (Davis et al., 1998) or by platelet-derived growth factor-induced activation of phosphatidyl inositol-3-kinase (Sims et al., 2000). The activities of glutamate transporters can also be regulated by protein-protein interactions. Glutamate transport associated protein for EAAT3 (GTRAP3–18) is a membrane-associated rat protein that interacts with the carboxy-terminal end of the rat neuronal glutamate transporter EAAT3 as shown by yeast two-hybrid analysis (Lin et al., 2001). GTRAP3–18 messenger RNA (mRNA) and protein are found in the same rat tissues and in the same regions of the rat brain as EAAT3. GTRAP3–18 reduces the affinity of EAAT3 for glutamate both in vitro and in vivo (Lin et al., 2001). GTRAP3–18, therefore, acts as an allosteric negative modulator of glutamate uptake via EAAT3. To date, no information has been provided on the mouse orthologue to GTRAP3–18. In the present study, we identified the murine orthologue of GTRAP3–18 and determined its gene structure as well as its tissue-specific expression profile. 2. Materials and methods 2.1. Isolation of total RNA Total RNA was isolated from mouse tissues using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer’s directions. 2.2. Isolation of mouse GTRAP3–18 DNA complementary to RNA (cDNA) The cDNA for mouse GTRAP3–18 was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers derived from the sequence of rat GTRAP3– 18 (GenBank number: AF240182). First-strand cDNA was synthesized from 5 mg adult C57bl/6J mouse brain total RNA using the SuperScript II reverse transcriptase (200 U; Invitrogen) according to manufacturer’s directions with the addition of RNase inhibitor (40 U; Roche Applied Science, Indianapolis, IN) and a GTRAP3–18-specific primer (2 pmol; GTRAP 1 567R; 5 0 -ttactccctggctttgctgatgtagtc-3 0 ). The first-strand cDNA was then amplified by PCR (30 cycles of 958C for 30 s, 608C for 30 s and 728C for 1 min followed by

728C for 10 min) using 500 nM of forward (GTRAP 1 1L; 5 0 -atggatgtgaaccttgccccgctccgtgc-3 0 ) and reverse primers, 1.7 U of Expand High Fidelity PCR System polymerase (Roche Applied Science), 200 mM dNTPs and Expand High Fidelity Buffer containing 2.5 mM MgCl2 in a PTC200 DNA Engine (MJ Research, Waltham, MA). PCR products were resolved on agarose gels via electrophoresis. PCR products were cloned into the pCR2.1 TOPO vector using the TOPO TA Cloning kit (Invitrogen) according to the manufacturer’s directions. Oligonucleotide primers for PCR were synthesized by Integrated DNA Technologies (Coralville, IA). 2.3. DNA sequencing and sequence analysis DNA sequencing reactions were conducted at the PlantMicrobe Genomics Facility of The Ohio State University using the Applied Biosystems 3700 DNA Analyzer with BigDye cycle sequencing terminator chemistry. Vector NTI Suite v.6 (InforMax, North Bethesda, MD) and algorithms available on the internet (CLUSTAL-W, NetPhos 2.0, NNSPLICE0.9, PhosphoBase, PROSITE, SEG, TBLASTN) were used to analyze mouse GTRAP3–18 DNA and amino acid sequences. 2.4. Isolation of the untranslated regions (UTRs) of mouse GTRAP3–18 RNA ligase-mediated rapid amplification of the cDNA 5 0 end (RLM-5 0 RACE; FirstChoice RLM-RACE kit, Ambion, Austin, TX) was used to isolate the 5 0 UTR of mouse GTRAP3–18 from brain and thymus total RNA according to the manufacturer’s directions. Nested PCR was performed with SuperTaq Plus (Ambion), 5 0 RACE adapter primers and gene-specific outer (GTRAP 1 223R; 5 0 -gcaccacaatgactcctccgaggatcat-3 0 ) and inner (GTRAP 1 187R; 5 0 ggctcagaaacccaacaaccgaaatc-3 0 ) primers. The 3 0 UTR of mouse GTRAP3–18 was isolated from brain and thymus total RNA using 3 0 RACE (FirstChoice RLM-RACE, Ambion) according to the manufacturer’s directions. Nested PCR was then performed with SuperTaq Plus (Ambion), 3 0 RACE adapter primers and gene-specific outer (GTRAP 1 342L; 5 0 -gatgttcgggggtgtcatggtctttg-3 0 ) and inner (GTRAP 1 368L; 5 0 -tgttcggcatcactcttcctttgctg3 0 ) primers. 2.5. Multiple tissue RT-PCR RT-PCR using total RNA isolated from mouse cerebral cortex, cerebellum, brainstem, spinal cord, skeletal muscle, heart, kidney and liver was performed as described in Section 2.2. First-strand cDNA libraries were generated as described in Section 2.2. GTRAP 1 1L and GTRAP 1 223 R primers were used to amplify mouse GTRAP3–18 transcripts as described in Section 2.2.

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

2.6. Antibody production Glutathione S-transferase (GST)-c-Myc-tagged rat GTRAP3–18 was used as the antigen for generating chicken anti-GTRAP3–18 protein antibodies. pGEX6P-1/GTRAP3– 18 (Lin et al., 2001) was transfected into Escherichia coli BL21 cells (Amersham Biosciences, Piscataway, NJ) according to previously described procedures. GST-cMyc-GTRAP3–18 fusion protein was isolated from the transformed BL21 cells according to established protocols (Frangioni and Neel, 1993). The immunization of chickens and subsequent harvesting of immunized eggs were conducted at Rockland Immunochemicals for Research, Inc. (Gilbertsville, PA). Two hens were immunized with 220 mg GST-GTRAP3–18 (dissolved in complete Fruend’s adjuvant) and were subsequently boosted with 100 mg antigen (dissolved in incomplete Fruend’s adjuvant) at days 5, 12 and 26. Eggs were harvested from the hens after it was determined that the test bleeds (from day 36) were immunoreactive to GTRAP3–18. Chicken IgY antibodies were purified from egg yolks using the EGGstract IgY Purification System (Promega, Madison, WI) according to manufacturer’s directions. Any antibodies that would have reacted to either the GST or the c-Myc moieties of the immunogen were removed from the antibody pool by affinity purification using a GST-c-Myc fusion protein. 2.7. Immunoblot analysis Tissues from adult mice or cell extracts were homogenized on ice in phosphate-buffer saline (PBS) containing Complete protease inhibitors (Roche Applied Science). A total of 20 mg of proteins were loaded onto a 10% polyacrylamide (PA) gel containing 0.1% sodium dodecylsulfate to be resolved by electrophoresis and then electrotransferred onto a polyvinylidene difluoride membrane (0.45 mm, Roche Applied Science) (Lin et al., 2001). These membranes were first incubated in blocking solution (5% non-fat milk in PBS 1 0.1% Tween-20) for 60 min at room temperature followed by primary antibody solution (either chicken anti-GTRAP3– 18 polyclonal antibody (pAb, 1:1500) or rabbit antiEAAT3 pAb (1:300; Rothstein et al., 1994) in 1% non-fat milk in PBS 1 0.1% Tween-20) overnight at 48C. After extensive washing, the membranes were incubated in secondary antibody solution (peroxidase-conjugated goat anti-chicken IgG (1:15,000; Rockland Immunochemicals for Research) or peroxidase-conjugated goat anti-rabbit IgG (1:1200; ICN Biomedicals, Aurora, OH) in 1% non-fat milk in PBS 1 0.1% Tween-20) for 60 min at room temperature. The immunoreactive bands were detected using the LumiLight Western Blotting Substrate (Roche Applied Science) according to manufacturer’s directions. 2.8. Immunohistochemistry Adult male C57bl/6J mice were perfused transcardially with 4% paraformaldehyde after being deeply anesthesized

83

with tribromoethanol (Avertin; 200 ml/10 g intraperitoneally). The brains were rapidly removed and cryoprotected with 15% sucrose for 24–48 h. Parasagittal sections (60 mm) were taken from the cryoprotected brains using a freezing microtome. Tissue sections were incubated in 0.3% H2O2 for 20 min at room temperature so as to remove any endogenous peroxidase activity in the tissues. After thorough washing, the sections were incubated in blocking solution (4% normal goat serum (NGS; Vector Laboratories, Burlingame, CA) and 0.1% Triton X-100 in Tris-buffered saline (TBS)) for 60 min at 48C and then in primary antibody solution (chicken anti-GTRAP3–18 pAb (1:3300) in 2% NGS and 0.1% Triton X-100 in TBS) overnight at 48C. After thorough washing with TBS, the sections were then incubated with biotinylated goat antichicken IgG (1:400; Vector Laboratories) in TBS containing 2% NGS for 60 min at 48C followed by thorough washing with TBS. The sections were then incubated with peroxidaseconjugated avidin (1:200; Vectastain Elite Standard kit, Vector Laboratories) for 60 min at 48C. Immunoreactivity was detected with Sigma Fast 3,3 0 -diaminobenzidine (DAB) tablets (Sigma-Aldrich, St. Louis, MO). After staining, the sections were dehydrated with graded ethanols, cleared with xylenes and coverslipped. All experiments were conducted in accordance with protocols described in the National Institute of Health Guide for the Care and Use of Animals and were approved by the local Institutional Laboratory Animal Care and Use Committee. All efforts were made to minimize the number of animals used and the suffering of these animals. 2.9. Generation of mammalian expression constructs and transient transfection Mouse GTRAP3–18 and mouse EAAT3 were subcloned into the pcDNA3 eukaryotic expression vector (Invitrogen). The complete open reading frame of mouse EAAT3 (GenBank number: NM_009199) was subcloned into the NotI and BamHI sites of pcDNA3 to form pcDNA3/ mEAAT3. pcDNA3/mGTRAP3–18 was generated by subcloning the complete open reading frame of mouse GTRAP3–18 into the NotI and BamHI sites of pcDNA3. Hep2G (generously provided by Arfaan Rampersaud) cells were transiently transfected with LipofectAMINE PLUS (Invitrogen) according to manufacturer’s directions. HEK293 (American Type Culture Collection, Manassas, VA) cells were transiently cotransfected with combinations of pcDNA3/mGTRAP3–18, pcDNA3/mEAAT3, pRK5/ GTRAP41 (Jackson et al., 2001) and pGL3Control (Promega) using LipofectAMINE PLUS. The pcDNA3 vector was also used in the cotransfection experiments. The amounts of total plasmid DNA, PLUS and LipofectAMINE used per well of a six-well plate were 1 mg, 6 and 4 ml for Hep2G cells and 2 mg, 14 and 4 ml for HEK293 cells. Cells were collected 72 h after transfection for analysis. Under these conditions, transfection efficiency is typically 80–90% (C.L.G. Lin, unpublished observations).

84

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

2.10. Glutamate uptake assay Uptake of radiolabeled glutamate was monitored in transfected cells as described previously (Lin et al., 2001). Briefly, cultured cells grown on six-well plates were washed with TS buffer (50 mM Tris–HCl and 320 mM sucrose, pH 7.4) and then incubated for 4 min at 378C with 2.04 nM l-[ 3H]glutamate (0.1 mCi, Amersham Biosciences, specific activity ¼ 49.0 mCi/nmol) in either Na 1-containing (120 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaCl2 and 555 mM d-glucose, pH 7.4) or Na 1-free (120 mM choline chloride, 25 mM Tris, 5 mM KCl, 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaCl2 and 555 mM dglucose, pH 7.4) Kreb’s buffer supplemented with 40 mM unlabeled glutamate. The cells were immediately placed on ice and washed three times with ice-cold PBS. The washed cells were then lysed in 1 mM NaOH and the amount of radiolabeled glutamate was measured using a Beckman Coulter LS6500 Multi-Purpose Scintillation Counter (Beckman Instruments, Fullerton, CA; counting efficiency for 3 H ¼ 68.0%). The amount of [ 3H]glutamate taken into the cells was calculated from the scintillation counting results using the following equation: nmol [ 3H]glutamate ¼ (counts per minute/efficiency)/(2.22 £ 10 6 disintegrations per minute/mCi)/(specific activity). Na 1-dependent glutamate uptake was obtained by subtracting the amount of glutamate incorporated into cells incubated with Na 1-free Kreb’s buffer from the amount of glutamate incorporated into cells incubated with Na 1-containing Kreb’s buffer.

3. Results and discussion 3.1. Molecular cloning and sequence analysis of mouse GTRAP3–18 We cloned the murine orthologue to GTRAP3–18 from adult cerebral cortex RNA by RT-PCR using primers spanning the coding region of rat GTRAP3–18 (Lin et al., 2001). The identity of the RT-PCR product was confirmed by Southern blot analysis using a probe derived from rat GTRAP3–18 cDNA (data not shown) and by sequencing. The 567-base pair open reading frame (ORF) for mouse GTRAP3–18 encoded a protein containing 188 amino acid residues (Fig. 1A; GenBank number AF421860). The mouse GTRAP3–18 ORF DNA sequence was 94.4% identical to the rat GTRAP3–18 DNA as determined by CLUSTAL-W alignment. The mouse GTRAP3–18 cDNA sequence was 96% identical to the cDNA encoding Aip-5 (Ingley et al., 1999). Aip-5 interacts with ARL-6, a novel member of the Ras superfamily of cytosolic GTP-binding proteins expressed in the brain, kidney and erythroid cells (Ingley et al., 1999). In order to obtain the complete cDNA of mouse GTRAP3– 18, we used RLM-5 0 RACE to obtain the 5 0 UTR and 3 0 -RACE for the 3 0 end of the gene. The 5 0 UTR of mouse GTRAP3–18

mRNA, which was determined by analysis of RLM-5 0 -RACE products from total RNA preparations from two different tissues, was 35 base pairs in length (Fig. 1A). There were no known regulatory domains found within the 5 0 UTR of mouse GTRAP3–18. The AUG start codon (136) is in very good context for translational initiation (Kozak, 1999). Mouse GTRAP3–18 mRNA contained a 733-bp 3 0 UTR as revealed from analysis of the 3 0 RACE products from two different RNA samples. The mouse GTRAP3–18 3 0 UTR contains one polyadenylation signal (AAUAAA) and five AUUU domains (Fig. 1A). These AUUU domains may be important in mRNA destabilization (Shaw and Kamen, 1986). The sequence of the 3 0 UTR obtained from 3 0 RACE strongly agreed with those from the full-length cDNAs found in the RIKEN Mouse Gene Encyclopedia Project (RIKEN Genome Exploration Research Group Phase II Team, FANTOM Consortium, 2001; GenBank numbers AK008519 and AK0052359) and the Mammalian Gene Collection (Strausberg et al., 1999; GenBank number BC003897). The apparent molecular mass of mouse GTRAP3–18 was 21.6 kDa. The predicted isoelectric point (pI) of mouse GTRAP3–18 was 9.62; mGTRAP3–18 would have a net charge of 15.94 at pH 7.0. PROSITE (Hofmann et al., 1999) and PhosphoBase (Kreegipuu et al. 1999) programs found two putative protein kinase C (PKC) phosphorylation sites (Fig. 1A, boxed with solid lines) as well as two putative cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation sites (Fig. 1A, boxed with dashed lines). Mouse GTRAP3–18 contained two serine residues (S18 and S30) and three tyrosine residues (Y42, Y47 and Y182; Fig. 1A, circled) that had a high probability of phosphorylation as determined using NetPhos 2.0 (Blom et al., 1999). S18 is one of the predicted PKC sites and S30 is one of the PKA/ CaMKII sites. These prediction algorithms suggest that GTRAP3–18 may be regulated by protein phosphorylation via intracellular signaling molecules. GTRAP3–18 was predicted to be a very hydrophobic protein as determined by Kyte and Doolittle hydropathy analysis (Fig. 1B) (Kyte and Doolitte, 1982). The secondary structure of mouse GTRAP3–18 was predicted to be composed of 62.2% ahelix and 8.0% b-sheet. However, there were no helices long enough to be transmembrane domains suggesting that GTRAP3–18 may be a membrane-associated protein. Mouse GTRAP3–18 protein was 97.9% identical to rat GTRAP3–18 and was 95.2% similar to human GTRAP3–18 (JWA) as determined by sequence alignments. Searching the non-mouse and non-human expressed sequence tag (EST) entries database with the translated BLAST algorithm (TBLASTN) (Altschul et al., 1997) produced nearly full-length amino acid sequences for the zebrafish (Danio rerio; AW305841 and AI877919) orthologue. Mouse GTRAP3–18 was 75.7% similar to the putative zebrafish protein. A search of the nr database using TBLASTN and the mouse GTRAP3–18 amino acid sequence produced two hypothetical proteins that were similar to mouse GTRAP3–

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

18: CG10373 from Drosophila melanogaster (43.6% similarity; AAF53696) and D2096.2 from Caenorhabditis elegans (48.3% similarity; AAA81495). The Fugu Genome database was searched using TBLASTN for the pufferfish (Fugu rubripes) orthologue. Pufferfish GTRAP3–18 was 72.0% similar to mouse GTRAP3–18. Alignment of these amino acid sequences showed two major regions that were conserved from nematode to man (Fig. 2A, boxed in black): LLY Y/FQTNY and N I/VHASLRLR. The second conserved domain contains one of the two putative PKC phosphoryla-

85

tion sites. The second conserved domain also contains part of a low complexity region as determined using the SEG algorithm (Wootton and Federhen, 1996). The aforementioned sequence alignment was then used to build an unrooted dendrogram (Fig. 2B) using the Neighbor Joining method of Saitou and Nei (1987). The branches of this unrooted dendrogram correspond closely to the taxonomic divisions of the species analyzed. The mammalian GTRAP3–18 proteins are closely related and probably derived from a single ancestor.

Fig. 1. Mouse GTRAP3–18 cDNA sequence. (A) Complete nucleotide sequence of mouse GTRAP3–18 (GenBank accession number AF421860). The predicted amino acid sequence is shown below its open reading frame. Putative PKC sites are boxed with solid lines and PKA/CaMKII sites are boxed with dashed lines. Putative phosphorylatable tyrosines are circled. The stop codon is boxed in grey. The polyadenylation signal is underlined and boldface. The AUUU regions of the 3 0 UTR are underlined. (B) Kyte-Doolittle hydropathy analysis of the predicted mouse GTRAP3–18 protein sequence.

86

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

Fig. 2. Evolutionary conservation of GTRAP3–18 protein. (A) CLUSTAL-W alignment of amino acid sequences from mouse, rat, human, pufferfish, zebrafish, fruitfly and nematode. Identical residues are blocked in black and similar residues are blocked in grey. GenBank accession numbers: human (XP_027135), rat (AAG28598), fruitfly (AAF53696) and nematode (AAA81495). The amino acid sequences for zebrafish and pufferfish GTRAP3–18s were derived from translated EST sequences (AI877919) and the Fugu Genome database, respectively. (B) Unrooted dendrogram derived from the CLUSTAL-W alignment using the Saitou-Nei Neighbor Joining method.

3.2. Genomic organization of mouse GTRAP3–18 We searched the Celera Mouse Genome Reference Database (Celera Discovery Systems) for genomic sequences containing fragments of GTRAP3–18 in order to determine the genomic organization of mouse GTRAP3–18. One genomic fragment from the database (Celera number GA_x5J8B7W7YD) showed significant homology to portions of the mouse GTRAP3–18 cDNA. This genomic fragment was then analyzed for splice donor and acceptor

sites using the Splice Site Prediction by Neural Network algorithm (NNSPLICE0.9; http://www.fruitfly.org/ seq_tools/splice.html). Splice donor and acceptor sites were indeed found within all of these fragments at the predicted sites. GTRAP3–18 gene contained three exons with two introns (Fig. 3). The exon-intron boundaries followed the 5 0 donor and 3 0 acceptor consensus sequences (GT…AG). The exon-intron organization of mouse GTRAP3–18 was confirmed by PCR (data not shown). In order to determine whether the genomic structure of

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

87

Fig. 3. Gene structure of mouse GTRAP3–18. Schematic diagram of the exon-intron organization of GTRAP3–18 in the mouse genome. The position of the translation start codon (ATG) within exon 1 and the translation stop codon within exon 3 are shown with lines.

GTRAP3–18 was conserved between mice and humans, we searched the Human Genome Database with BLASTN for genomic sequences containing GTRAP3–18.The exonintron organization of the human GTRAP3–18 gene is identical to the mouse gene with respect to exon sizes and locations (International Human Genome Sequencing Consortium, 2001; Venter et al., 2001). The sizes of the two human GTRAP3–18 introns (15,079 nt for intron 1 and 2408 nt for intron 2) were found to be similar to those of the murine orthologue. We also determined the genomic organization of GTRAP3–18 from the pufferfish F. rubripes. The Fugu Genome database revealed that pufferfish GTRAP3–18 was divided into three exons. The exonintron boundaries for pufferfish GTRAP3–18 were in the same positions as those for mouse and human GTRAP3– 18s. Interestingly, the sizes of the two pufferfish GTRAP3–18 introns were significantly smaller (910 nt for intron 1 and 247 nt for intron 2) than the sizes of the mouse or human introns. The sizes of pufferfish introns are typically smaller those from humans or mice since the overall size of the pufferfish genome is smaller than the human genome (Brenner et al., 1993). This similarity in genome structure between mice, humans and pufferfish suggests that the organization of GTRAP3–18 is evolutionarily conserved amongst the vertebrates. Databases from the National Center for Biotechnology Information (LocusLink) and from Celera Discovery Systems have placed GTRAP3–18 (Arl6ip5) on mouse chromosome 6D3. Interestingly, Tmf1 (TATA element modulatory factor 1), Lmod2 (cardiac leiomodin) and Golgi-associated band 4.1like protein are three genes located in close proximity to GTRAP3–18 that encode cytoskeletal proteins. Preliminary results (J.W. Zhou, Y.P. Di and R. Wu, unpublished observations) suggest that human GTRAP3–18 protein may be associated with the cytoskeleton. If GTRAP3–18 is, indeed, associated with the cytoskeleton, then the close proximity of GTRAP3–18, Tmf1, Lmod2 and Golgi-associated band 4.1like protein could be an example of a genomic clustering of genes with a common function. This region of mouse chromosome 6D3 is syntenic with human chromosome 3p14. The short arm of chromosome 3 has been implicated in various adult cancers of the kidney, lung, testes and mammary tissue (Kok et al., 1997). Interestingly, linkage analysis suggests that

3p14 is a susceptibility locus for generalized idiopathic epilepsy (Zara et al., 1998). The Fugu Genome database revealed that the gene for ubiquitin-activating enzyme E1C (UBE1C) was adjacent to GTRAP3–18 in the pufferfish genome. UBE1C was also adjacent to GTRAP3–18 in both the mouse and human genomes suggesting a high level of synteny between pufferfish, mouse and human genomes (Brunner et al., 1999). 3.3. Expression profile of GTRAP3–18 RNA and protein in mouse tissues RT-PCR was used to examine the expression profile of GTRAP3–18 RNA in mouse tissues. Mouse GTRAP3–18 RNA was found in every tissue examined including the brain, heart, liver, skeletal muscle and kidney (Fig. 4A). The PCR products shown in Fig. 4A were not the result of amplification of contaminating genomic DNA since the PCR primers used contain exon 2 sequences. The mouse EST database contained 118 GTRAP3–18-containing sequences. These ESTs were derived from cDNA libraries generated from a variety of tissue sources including brain, olfactory bulb, cerebellum, kidney, muscle, lung, retina, thymus, spleen, mammary gland, salivary gland, pituitary gland, macrophages, germinal B-cells and T cells. The RTPCR data along with the EST database show that GTRAP3– 18 RNA is found in every tissue of the mouse. The expression profile of mouse GTRAP3–18 RNA is very similar to that reported for EAAT3 (Mukainaka et al., 1995). The distribution of GTRAP3–18 protein in different mouse tissues was examined by immunoblot analysis using an antiGTRAP3–18 polyclonal antibody generated in chickens. This chicken polyclonal antibody is unique in that it was generated against the entire GTRAP3–18 protein instead of a particular polypeptide epitope. This chicken polyclonal antibody reacts with its antigen (purified GST-tagged c-Myc-GTRAP3–18) as well as mouse GTRAP3–18 ectopically expressed in Hep2G cells (Fig. 4B). Preincubation of the chicken antibody with its antigen prevented binding to GTRAP3–18 protein present in a mouse brain extract (Fig. 4B, lane Ag block) indicating that this antibody reacted specifically with GTRAP3–18. GTRAP3–18 protein was expressed in all of the adult mouse tissues tested including the brain, heart, liver and kidney (Fig.

88

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

Fig. 4. GTRAP3–18 RNA and protein expression profiles in mouse tissues. (A) GTRAP3–18 RNA expression profile in adult mouse tissues using RT-PCR. A 223-bp GTRAP3–18 PCR product was observed in the nervous system as well as in peripheral tissues such as the kidney, liver, heart and skeletal muscle. Omission of first-strand cDNA served as a negative control for PCR. (B) GTRAP3–18 protein expression profile in adult mouse tissues as shown by immunoblot analysis. The chicken anti-GTRAP3–18 polyclonal antibody reacted to purified GST-tagged c-Myc-GTRAP3–18 as well as GTRAP3–18 ectopically expressed in Hep2G cells; no immunoreactive bands were observed when a mouse brain extract was incubated with GST-c-Myc-GTRAP3–18blocked antibody (Ag block lane). Antibodies from preimmune chickens (pre IgY lane) did not react to any proteins in mouse brain extracts. The chicken antibody, therefore, specifically reacted to GTRAP3–18. GTRAP3–18 monomers, dimers, trimers are shown by arrowheads, arrows and open arrows, respectively; multiple bands within the GTRAP3–18 dimers are shown by an asterisk. Ectopically expressed mouse GTRAP3–18 tended to migrate as a trimer (in Hep2G as well as HEK293 (not shown) cells) although GTRAP3–18 dimers and monomers were observed upon prolonged exposure. (C) GTRAP3– 18 protein expression within the adult mouse brain. GTRAP3–18 was observed in neurons of paraformaldehyde-fixed mouse brain sections incubated with the chicken anti-GTRAP3–18 antibody. As a negative control, immunohistochemistry was performed in the absence of primary antibody (data not shown). GTRAP3–18 immunoreactivity was found in the cerebral cortex, striatum, hippocampus and cerebellum. Strong GTRAP3–18 immunoreactivity was observed in the neuron-rich stratum pyrimidale of the hippocampus and the Purkinje cells of the cerebellum. The scale bar on each picture represents 10 mm.

4B). The expression profile of GTRAP3–18 protein was consistent with that of GTRAP3–18 RNA as shown by RTPCR. GTRAP3–18 tended to form dimers, trimers and other higher order multimers even under reducing conditions (Fig. 4B). This oligomerization was probably a consequence of the hydrophobic nature of the protein. A lower mouse GTRAP3– 18 dimer band (asterisk) was consistently observed in homogenates of mouse brain, liver and skeletal muscle. This observation could be the result of multiple GTRAP3–18 isoforms, post-translational modifications of GTRAP3–18 protein or polyspecific nature of the anti-GTRAP3–18 antibody. The oligomerization and multiple band patterns that were observed using the chicken anti-GTRAP3–18 protein antibody were similar to those observed using different rabbit antiGTRAP3–18 peptide antibodies (unpublished observation). Expression of GTRAP3–18 protein within specific

regions of the mouse brain was determined by immunohistochemistry. There was strong GTRAP3–18 immunoreactivity in the cerebral cortex, hippocampus, striatum and cerebellum (Fig. 4C). GTRAP3–18 immunoreactivity was localized primarily within the stratum pyramidale of the hippocampus, an area heavily populated with neurons. Within the cerebellum, the immunoreactivity was primarily seen in the Purkinje cell layer and the granular layer. The amount of staining was very low when brain sections were incubated without the primary antibody (data not shown) thereby showing the observed immunoreactivity was specific to GTRAP3–18. GTRAP3–18 protein is expressed in the same brain regions as EAAT3 (Furuta et al., 1997). Interestingly, GTRAP3–18 protein is expressed in brain regions that contain high densities of glutamatergic and GABAergic neurons.

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

3.4. Mouse GTRAP3–18 regulates EAAT3-mediated glutamate uptake Rat GTRAP3–18 negatively modulates EAAT3mediated glutamate transport (Lin et al., 2001). In order to determine the function of mouse GTRAP3–18, we transiently transfected HEK293 cells with different amounts of mouse GTRAP3–18 but the same amount of mouse EAAT3. Na 1-dependent glutamate uptake was then measured in these transfected cells (Fig. 5A). Na 1-independent glutamate uptake contributed to less than 10% of the total glutamate uptake under the experimental conditions used in this study (3.78 ^ 0.32 fmol [ 3H]glutamate/ml lysate). Mouse GTRAP3–18 inhibited EAAT3-mediated Na 1-dependent glutamate transport in a dose-dependent manner. In fact, Na 1-dependent glutamate uptake was reduced by 42% when equivalent amounts of GTRAP3–18 and EAAT3 were present in transfected cells. Mouse GTRAP3–18 has no effect on Na 1-independent glutamate uptake under the experimental conditions used in this study. The expression of mouse GTRAP3–18 protein increased with increasing

Fig. 5. Mouse GTRAP3–18 negatively modulates Na 1-dependent, EAAT3mediated glutamate uptake. Glutamate uptake assays were performed on HEK293 cells transiently transfected with either mouse EAAT3, mouse GTRAP3–18, GTRAP41 or luciferase cDNAs. The quantities of plasmid DNAs used for each condition are given below the bar graph. The amount of [ 3H]glutamate taken into the transfected cells in a Na 1-dependent manner is shown as mean glutamate uptake ^ SEM (n ¼ 4). Na 1-independent glutamate uptake contributed to less than 10% of the total glutamate uptake (3.78 ^ 0.32 fmol [ 3H]glutamate/ml lysate) and not altered by the expression of mouse GTRAP3–18. Mouse GTRAP3–18 inhibited mouse EAAT3-mediated glutamate transport in a dose-dependent manner. The expression of mouse GTRAP3–18 protein increased with amount of mouse GTRAP3–18 plasmid DNA while the expression of mouse EAAT3 protein was the same in all samples. Coexpression of GTRAP41 or luciferase with mouse EAAT3 had no effect on Na 1-dependent [ 3H]glutamate uptake.

89

amounts of transfected GTRAP3–18-containing plasmid DNA while the expression of mouse EAAT3 protein was the same in all samples. GTRAP3–18, therefore, functions as a negative modulator of EAAT3-mediated glutamate uptake. To demonstrate that the modulation of EAAT3mediated glutamate uptake was specific for GTRAP3–18, glutamate uptake was measured in HEK293 cells cotransfected with mouse EAAT3 cDNA and either GTRAP41 or luciferase cDNAs. GTRAP41 negatively modulates EAAT4-mediated glutamate uptake but has no effect on EAAT3-mediated glutamate uptake (Jackson et al., 2001). The amounts of [ 3H]glutamate taken into EAAT3/ GTRAP41- and EAAT3/luciferase-expressing HEK293 cells were similar to the amount taken into EAAT3-expressing cells (Fig. 5A). These results show that reduction of EAAT3-mediated glutamate transport by GTRAP3–18 is specific for GTRAP3–18. 3.5. Conclusions We have isolated and characterized the murine orthologue to GTRAP3–18, a novel protein that regulates glutamate transport across the plasma membrane. Mouse GTRAP3–18 protein is a hydrophobic protein that tends to form homomultimers even under reducing conditions. There are putative serine and tyrosine phosphorylation sites within the GTRAP3–18 amino acid sequence. GTRAP3–18 is a highly conserved protein amongst mammals (human, mouse and rat) and orthologues exist in zebrafish, pufferfish, fruit flies and nematodes. The GTRAP3–18 gene is divided into three exons and is separated by two introns; the genomic structure of GTRAP3–18 is very similar between mice, humans and pufferfish. GTRAP3–18 is located on mouse chromosome 6D3, a region that is syntenic to human chromosome 3p14 – a region that contains susceptibility loci for generalized seizures and for cancer. GTRAP3–18 mRNA and protein are expressed in the central nervous system as well as peripheral organs such as the kidney and liver. GTRAP3– 18 protein is localized in neuron-rich regions within the mouse brain such as the stratum pyramidale of the hippocampus and the Purkinje cells of the cerebellum. The expression profile of GTRAP3–18 is very similar to that of its interacting partner EAAT3. Mouse GTRAP3–18 negatively modulates mouse EAAT3-mediated glutamate uptake in a dose-dependent manner. This information will allow us to further investigate the biology of GTRAP3–18 and its importance in regulating glutamate uptake into cells. Acknowledgements We would like to thank Dr Georgia Bishop (Department of Neuroscience) for providing access to the freezing microtome as well as advise with regard to immunohistochemistry, Dr Richard Burry (Department of Neuroscience) and the Campus Microscopy Imaging Facility for assistance with

90

M.E.R. Butchbach et al. / Gene 292 (2002) 81–90

image acquisition, Dr Arfaan Rampersaud (Division of Neuropathology) for providing the Hep2G cells, Dr Jeffrey Rothstein (Department of Neurology, John Hopkins University) for generously providing the EAAT3 pAb and David Brochu (Rockland Immunochemicals for Research, Inc.) for his assistance in generating the chicken anti-GTRAP3–18 antibody. Mouse genomic sequence data were obtained through the use of the Celera Discovery System and its associated databases. Pufferfish genomic sequence data has been provided freely by the Fugu Genome Consortium for use in this publication only. M. E. R. B. was supported in part by a predoctoral research fellowship from the Epilepsy Foundation of America and the American Epilepsy Society. References Altschul, S.F., Madden, T.L., Scha¨ ffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Blom, N., Gammeltoft, S., Brunak, S., 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, 1351–1362. Brenner, S., Elgar, G., Sandford, R., Macrae, A., Venkatesh, B., Aparicio, S., 1993. Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome. Nature 366, 265–268. Brunner, B., Todt, T., Lenzner, S., Stout, K., Schulz, U., Ropers, H.H., Kalscheuer, V.M., 1999. Genomic structure and comparative analysis of nine Fugu genes: conservation of synteny with human chromosome Xp22.2-p22.1. Genome Res. 9, 437–448. Coco, S., Verderio, C., Trotti, D., Rothstein, J.D., Volterra, A., Matteoli, M., 1997. Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons. Eur. J. Neurosci. 9, 1902– 1910. Conti, F., DeBiasi, S., Minelli, A., Rothstein, J.D., Melone, M., 1998. EAAC1, a high-affinity glutamate transporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex. Cereb. Cortex 8, 108–116. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Davis, K.E., Straff, D.J., Weinstein, E.A., Bannerman, P.G., Correale, D.M., Rothstein, J.D., Robinson, M.B., 1998. Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma. J. Neurosci. 18, 2475–2485. Eccles, C.U., Dykes-Hoberg, M., Rothstein, J.D., 1996. Inhibition of synthesis of EAAC1 glutamate transporter alters g-aminobutyric acid levels in discrete brain regions. Soc. Neurosci. Abs. 22, 1570. Frangioni, J.V., Neel, B.G., 1993. Solubilization and purification of enzymatically active glutathione-S-transferase (pGEX) fusion proteins. Anal. Biochem. 210, 179–187. Furuta, A., Martin, L.J., Lin, C.L.G., Dykes-Hoberg, M., Rothstein, J.D., 1997. Cellular and synaptic localization of the neuronal glutamate transporters excitatory amino acid transporter 3 and 4. Neuroscience 81, 1031–1042. Hofmann, K., Bucher, P., Falquet, L., Bairoch, A., 1999. The PROSITE database, its status in 1999. Nucleic Acids Res. 27, 215–219. Ingley, E., Willians, J.H., Walker, C.E., Tsai, S., Colley, S., Sayer, M.S., Tilbrook, P.A., Sarna, M., Beaumont, J.G., Klinken, S.P., 1999. A novel

ADP-ribosylation like factor (ARL-6) interacts with the proteinconducting channel SEC61b subunit. FEBS Lett. 459, 69–74. International Human Genome Sequencing Consortium, 2001. Initial sequencing and analysis of the human genome. Nature 409, 860–921. Jackson, M., Song, W., Liu, M.Y., Jin, L., Dykes-Hoberg, M., Lin, C.L.G., Bowers, W.J., Federoff, H.J., Sternweis, R.C., Rothstein, J.D., 2001. Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins. Nature 410, 89–93. King, N., Williams, H., McGivan, J., Suleiman, M.S., 2001. Characteristics of l-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart. Cardiovasc. Res. 52, 84–94. Kok, K., Naylor, S.L., Buys, C.H.C.M., 1997. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv. Cancer Res. 71, 27–92. Kozak, M., 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208. Kreegipuu, A., Blom, N., Brunak, S., 1999. PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res. 27, 237–239. Kyte, J., Doolitte, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. Lin, C.L.G., Orlov, I., Ruggerio, A.M., Dykes-Hoberg, M., Lee, A., Jackson, M., Rothstein, J.D., 2001. Modulation of the neuronal glutamate transporter EAAC1 by an interacting protein GTRAP3–18. Nature 410, 84–88. Mukainaka, Y., Tanaka, K., Hagiwara, T., Wada, K., 1995. Molecular cloning of two glutamate transporter subtypes from mouse brain. Biochim. Biophys. Acta 1244, 233–237. Peghini, R., Janzen, J., Stoffel, W., 1997. Glutamate transporter EAAC-1deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J. 16, 3822–3832. RIKEN Genome Exploration Research Group Phase II Team, FANTOM Consortium, 2001. Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690. Rothstein, J.D., Martin, L., Levey, A.I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N., Kuncl, R.W., 1994. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725. Rothstein, J.D., Dykes-Hoberg, M., Pardo, C.A., Bristol, L.A., Jin, L., Kuncl, R.W., Kanai, Y., Hediger, M.A., Wang, Y., Schielke, J.P., Welty, D.F., 1996. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Shaw, G., Kamen, R., 1986. A conserved AU sequence from the 3 0 untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659–667. Shayakul, C., Kanai, Y., Lee, W.S., Brown, D., Rothstein, J.D., Hediger, M.A., 1999. Localization of the high-affinity glutamate transporter EAAC1 in rat kidney. Am. J. Physiol. 273, F1023–F1029. Sims, K.D., Straff, D.J., Robinson, M.B., 2000. Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase. J. Biol. Chem. 275, 5228–5237. Strausberg, R.L., Feingold, E.A., Klausner, R.D., Collins, F.S., 1999. The mammalian gene collection. Science 286, 455–457. Venter, J.C., et al., 2001. The sequence of the human genome. Science 291, 1304–1351. Wootton, J.C., Federhen, S., 1996. Analysis of compositionally biased regions in sequence databases. Methods Enzymol. 266, 554–571. Zara, F., Labuda, M., Gaetano Garofalo, P., Durisotti, C., Bianchi, A., Castellotti, B., Patel, P.I., Avanzini, G., Pandolfo, M., 1998. Unusual EEG pattern linked to chromosome 3p in a family with idiopathic generalized epilepsy. Neurology 51, 493–498.