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11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
Received for publication September 14, 2007.
Accepted for publication April 14, 2008.
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11) missing the last three transmembrane domains of the full-length mENT1. The mENT111 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT111 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT111 in terms of transport function or inhibitor binding affinity. PK15-mENT111 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT111 could not be photolabeled with [3H]NBMPR and that mENT111 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.
; King et al., 2006; Zhang et al., 2007). In most cell types, this occurs by facilitative diffusion through a family of equilibrative nucleoside transporters (ENTs) (Hyde et al., 2001). The best characterized of this family is ENT1, which is distinguished functionally by its high affinity for the inhibitor nitrobenzylmercaptopurine riboside (NBMPR; nitrobenzylthioinosine) (Hyde et al., 2001). ENT1 mediates the bidirectional transport of a variety of purine and pyrimidine nucleosides, including adenosine, which is known to have endogenous cardiovascular and neural regulatory functions. The ENT1 gene was identified in human and rat tissues in 1997 (Coe et al., 1997; Griffiths et al., 1997; Yao et al., 1997), and we obtained the cDNA for the mouse ENT1 in 2000 (Kiss et al., 2000). The protein encoded by mENT1 is predicted to consist of 458 amino acids in an 11-transmembrane domain (TM) topology with a large glycosylated extracellular loop joining TMs 1 and 2, and a large intracellular loop between TMs 6 and 7 (Fig. 1). The central intracellular loop is predicted to contain phosphorylation sites for casein kinase II (CK2) and protein kinase C. We, and others, have identified an alternative splice variant of mENT1 that is missing a Lys-Gly in the central intracellular loop and has an arginine in place of a serine in the position (254) preceding the deletion (mENT1a; mENT1.2) (Kiss et al., 2000; Handa et al., 2001). This results in the loss of a potential CK2 phosphorylation site. We have shown that this shorter variant differs from the full-length mENT1 in that it is insensitive to the transporter-modifying effects of CK2 inhibitors and has a significantly higher affinity for [3H]NBMPR (Bone et al., 2007).
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We have now identified a third mENT1 variant that arises from the skipping of exon 11 during preRNA processing, designated mENT111 (GenBank accession no. EU180577
[GenBank]
). This variant was first noted as a 1242-bp RT-PCR product from mouse skeletal muscle when using primers to the open reading frame of mENT1 (1500 bp; GenBank accession no. AF131212.1). In silica translation of the sequence indicated that the protein encoded by this mRNA would be lacking 102 amino acids from the C terminus as a result of the introduction of a frame-shift resulting in a stop codon after position 356. Preliminary studies, presented at the XVth World Congress of Pharmacology (Robillard et al., 2006), showed that mENT111 encoded a protein that retained high affinity for [3H]NBMPR. The present study reports on the complete characterization of this novel splice variant.
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Materials and Methods |
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Escherichia coli, the Benchmark Prestained Protein Ladder, and the pcDNA3.1TOPO kit were purchased from Invitrogen (Burlington, ON, Canada). Mini-prep Plasmid DNA kits were obtained from QIAGEN (Mississauga, ON, Canada), and cloning rings were purchased from Bel-Art Scienceware (Pequannock, New Jersey, USA). U2-osteosarcoma (U2-OS) cells were a gift from Dr David Litchfield (University of Western Ontario, London, ON, Canada) and the PK15-NTD (nucleoside transport deficient) cells used for creating the stable mENT1 cell lines were provided by Dr. Ming Tse (Johns Hopkins University, Baltimore, MD).
Plasmid Generation. mENT1 and mENT111 were obtained from mouse skeletal muscle tissue by RT-PCR using Platinum Taq polymerase and primers complementary to the 5' and 3' termini of the open reading frame of mENT1 (5'mENT1, 3'mENT1-Kpn1; Table 1). PCR conditions were as follows: initial activation for 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, and a final 10-min elongation at 72°C. The PCR products were ligated into the multiple cloning site of pcDNA3.1 using the TOPO cloning procedure. All constructs were sequenced in both directions (Robarts Research Institute, Sequencing Facility, London, ON, Canada) using the Taq BigDye Terminator Cycle Sequencing kit in an automated ABI PRISM model 377 Version 3.3 DNA sequencer (Applied Biosystems, Streetsville, ON, Canada). The nucleotide sequences were translated and transmembrane topology predictions were made using the Tmpred software (http://www.ch.embnet.org/software/TMPRED_form.html).
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To generate plasmids encoding mENT1 or mENT111 with an N-terminal FLAG epitope tag (DYKYYYD), pcDNA3.1-mENT1 and pcDNA3.1-mENT111 were used as templates for PCR with primers containing the appropriate restriction enzyme cut sites (5'HindIII-mENT1 and 3'mENT1-Kpn1; Table 1) for ligation into the p3xFLAG-CMV10 vector. PCR products were separated on 1.2% agarose gels containing ethidium bromide, cut with the appropriate restriction enzyme, and ligated into p3xFLAG-CMV10. DH5 sub-cloning efficiency E. coli cells were transformed with the plasmid constructs using 42°C heat shock according to manufacturer's protocol (Invitrogen) and plated on Luria-Bertani broth-agar plates containing 100 µg/ml ampicillin. Plasmid DNA was isolated from transformed DH5 cells according to manufacturer's protocols (QIAGEN).
Stable Transfections. PK15-NTD cells were transfected with pcDNA3.1-mENT1, pcDNA3.1-mENT111, p3xFLAG-CMV10-mENT1, or p3xFLAG-CMV10-mENT111 using Lipofectamine 2000. The ratio of DNA to Lipofectamine 2000 was 1:3, using approximately 4.0 µg of plasmid DNA. Transfected cells were selected based on survival in 500 µg/ml G418, and individual cell colonies were isolated after limiting dilution of the surviving cells. The PK15-mENT1 and PK15-mENT111 cell lines were maintained at 37°C in a5%CO2 humidified atmosphere in vented tissue culture flasks containing modified Eagle's medium supplemented with 10% (v/v) bovine growth serum (BGS), 100 units of penicillin, 100 µg/ml of streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 300 µg/ml G418. mRNA was collected from each cell clone and tested for the presence of the respective mENT1 or mENT111 transcript by RT-PCR using primers spanning the open reading frame of mENT1 (Table 1).
Immunoblotting of Transiently Transfected U2-OS Cells. Transient transfection of U2-OS cells with p3xFLAG-mENT1 and p3xFLAG-mENT111 was conducted to confirm that the encoded proteins were being properly expressed. U2-OS cells were grown in Dulbecco's modified Eagle's medium with 10% BGS (v/v), 100 units of penicillin and 100 µg/ml streptomycin. p3xFLAG-mENT1, p3xFLAG-mENT111, or empty p3xFLAG-vector (25 µg/175-cm2 flask) was diluted in 10 mM Tris-HCl and 1 mM EDTA, pH 7.3, containing 0.25 mM CaCl2. This DNA+CaCl2 solution was then added drop wise to bubbling 2x concentrated HEPES-buffered saline (280 mM NaCl, 50 mM HEPES, and 1.5 mM Na2HPO4, pH 7.05). The mixture was incubated at room temperature for 20 min to form a calcium precipitate and was then added to 40% confluent U2-OS cells in a 175-cm2 flask. The cells were incubated with the precipitate overnight (
16 h) at 37°C in a 5% CO2 atmosphere, then washed with PBS and fresh media was added. Cells were harvested 48 h after initial transfection for the preparation of cell membranes.
U2-OS cells expressing p3xFLAG-mENT1, p3xFLAG-mENT111, or empty vector were removed from their flasks with 0.05% Trypsin/EDTA and resuspended in 5 mM sodium phosphate buffer (5 mM Na2HPO4, pH 7.2) containing a mammalian protease inhibitor cocktail. Cells were incubated in the lysis buffer for 30 min on ice and were subjected to sonication using a Sonic Dismembrator model 150 (Thermo Fisher Scientific, Waltham, MA) (setting 5, 30 s x 3). Cell/membrane suspensions were then centrifuged (4°C, 3000g x 30 min) to pellet nuclei and unbroken cells. The supernatant was centrifuged (4°C) for 1 h at 30,000g, and the pelleted membranes were suspended in 5 mM sodium phosphate lysis buffer containing protease inhibitor cocktail. Bradford colormetric protein assays (Thermo Fisher Scientific, Waltham, MA) were performed on each membrane preparation to quantify total protein yield.
For immunoblotting, 20 µg of membrane protein was denatured for 2 min at 100°C in SDS sample buffer (0.5M Tris-Cl, pH 6.8, 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.0012% bromphenol blue) and subjected to SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel. Samples were then transferred to polyvinylidene difluoride (PVDF) membranes using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA). The PVDF membranes were blocked overnight with 5% skim milk in TBS-T buffer (0.5 mM Tris, 13.8 mM NaCl, 2.7 mM KCl, and 0.01% Tween 20). After 3 washes of 10 min each in fresh TBS-T buffer, PVDF membranes were incubated with primary polyclonal rabbit anti-FLAG antibody for 2 h at room temperature (1:1000 dilution, 3% skim milk in TBS-T buffer). After three washes of 10 min each with TBS-T buffer, PVDF membranes were incubated with polyclonal goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000 dilution, 3% skim milk in TBS-T buffer) for 1 h at room temperature. Antigen reactivity was detected using LumiGLO Chemiluminescent Substrate (Millipore Bioscience Research Reagents, Temecula, CA). Molecular mass was determined by comparing Rf values against a Benchmark Prestained Protein Ladder.
[3H]NBMPR Binding. Cells were removed from flasks by trypsinization [0.05% (v/v) trypsin and 0.53 mM EDTA, 5 min, 37°C] and diluted with their respective media containing 10% (v/v) BGS and collected by centrifugation. Cell pellets were washed once by resuspension/centrifugation in isotonic N-methyl-glucamine (NMG) buffer, pH 7.25, containing 140 mM NMG, 5 mM KCl, 4.2 mM KHCO3, 0.36 mM K2HPO4, 0.44 mM KH2PO4, 10 mM HEPES (sodium free), 0.5 mM MgCl2, and 1.3 mM CaCl2 and then resuspended in this buffer for subsequent assays. Cell concentrations were determined routinely using a hemocytometer. In some cases, cells were incubated for 30 min on ice with 300 µM NEM, and then washed four times with NMG buffer to remove unreacted NEM before use in the binding assays.
PK15-NTD, PK15-mENT1, or PK15-mENT111 (± 3xFLAG) cells, and isolated membranes prepared as described above, were incubated with [3H]NBMPR using procedures that we have described previously (Bone et al., 2007). Nonspecific binding of [3H]-NBMPR was defined as that seen in the presence of 10 µM NBTGR. This concentration of NBTGR is 1000-fold greater than its KI for inhibition of [3H]NBMPR binding to ENT1 (Hammond, 1991). Specific binding of [3H]NBMPR was calculated as total minus nonspecific binding. Nonlinear regression analysis (Prism ver. 4.0; Graph-Pad Software, San Diego, CA) was used to fit rectangular hyperbolic curves to the site-specific binding of [3H]NBMPR plotted against the free [3H]NBMPR concentration at steady-state.
[3H]NBMPR Photoaffinity Labeling. Membranes prepared from PK15-mENT1 and PK15-mENT111 cells were photolabeled with [3H]NBMPR as we have described previously (Hammond and Johnstone, 1989). In brief, membranes were incubated with 5 nM [3H]NBMPR for 40 min at room temperature to allow for steady-state binding. Dithiothreitol (10 mM final conc.) was then added and the mixture was placed in a Petri dish on ice and exposed to 45 s of UV light (repeated 3 times with 1 min cooling on ice between UV exposures) from a 200-W mercury-arc lamp at a distance of 4 cm, with constant stirring with a magnetic stir bar. Membranes were washed three times in 5 mM sodium phosphate buffer by centrifugation at 10,000g for 3 min, and suspended in Laemmli's SDS-PAGE buffer and incubated at 50°C for 5 min to denature protein. Solubilized membranes were electrophoresed on a 6% SDS-polyacrylamide stacking gel and 5 to 15% gradient SDS-polyacrylamide separating gel. Gels were then fixed in 5% acetic acid, and each gel lane was cut into 2-mm slices for analysis of [3H]content. Radioactivity was plotted against the distance traveled (mm) in the gel and was compared with the reference protein ladder to determine approximate molecular mass of the radiolabeled proteins.
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11 and mENT1, [3H]2-chloroadenosine and [3H]uridine uptake was examined using methods described previous by our laboratory (Stolk et al., 2005; Bone et al., 2007). In brief, cells were incubated with [3H]substrate (± test inhibitors) in 1.5-ml microcentrifuge tubes over a 200-µl layer of 84% silicone/16% light mineral oil. Uptake reactions were terminated by centrifugation of the cells through the oil layer followed by digestion of the cell pellet in 1 M NaOH. Transporter-mediated uptake was defined as the total cell accumulation of radiolabel minus the amount of cell associated radiolabel in the presence of a supramaximal inhibitory concentration (10 µM) of the ENT inhibitors NBTGR and dipyridamole. In some cases, cells were incubated with 300 µM NEM for 30 min on ice and then washed extensively with NMG buffer (4 times) before assessment of [3H]2-chloroadenosine uptake.
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11. To examine the relative endogenous distribution of mRNA transcript and protein corresponding to mENT1 and mENT111, a range of tissues (heart, lungs, liver, kidney, brain, pancreas, thymus, skeletal muscle, spleen, and testes) from three male C57BL/6 mice were isolated and either digested in guanidinium isothiocyanate buffer (4.2 M, containing 25 mM sodium citrate, 1 mM EDTA, and 0.007% β-mercaptoethanol, pH 7.0) for RNA extraction, or snap-frozen in liquid nitrogen for subsequent immunoblotting. Total RNA was isolated using standard phenol-chloroform extraction methods, and mRNA was purified from total RNA using the Sigma mRNA purification kit. cDNA was generated from isolated mRNA, then target transcripts were amplified using Platinum Taq polymerase and primers spanning the full coding region of mENT1 (5'mENT1 and 3'mENT1-Kpn1; Table 1). The 3' primer is designed to anneal to a region located near the end of exon 12, which is present in both the mENT1 and mENT111, allowing amplification of both transcripts within a single reaction. PCR products were then electrophoresed on a 2% agarose gel with ethidium bromide for approximately 1 h (100 mV, room temperature). Gels were photographed on a digital Alpha Innotech (San Leandro, CA) imaging system.
For immunoblotting, frozen tissue was homogenized in 3 ml/g radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail. Protein samples (100 µg) were denatured for 2 min at 100°C in SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis on a 12.5% gel. Samples were then transferred to PVDF membranes, blocked with 5% skim milk in TBS-T for 2 h at room temperature, then immunolabeled with goat anti-ENT1 polyclonal antibody diluted 1:200 in blocking solution at 4°C overnight (16 h). Membranes were washed in TBS-T and subsequently incubated with donkey anti-goat secondary antibody conjugated to horseradish peroxidase (1:2000 dilution) for 2 h at room temperature. After an additional wash, immunoreactivity was detected using LumiGLO Chemiluminescent Substrate. Molecular mass was determined by comparing Rf values against a Benchmark Prestained Protein Ladder. Specificity of the ENT1 antibody was confirmed by its lack of immunoreactivity to membranes prepared from PK15NTD cells that do not express ENT1.
To investigate the existence of a human ENT1 splice variant analogous to mENT111, human major tissue cDNA I and II (Bio-chain Institute, Hayward, CA) isolated from brain, heart, kidney, liver, lung, pancreas, placenta, spleen, and skeletal muscle were screened by PCR, using the conditions described above, with primers designed to span the open reading frame of hENT1 (5'hENT1 and 3'hENT1;Table 1). mRNA was also isolated from human umbilical microvascular endothelial cells and human U2-OS cells and screened for ENT111 like transcripts as described above.
Statistical and Data Analyses. All data were analyzed using Lotus 1-2-3 1997 and [3H]NBMPR and [3H]substrate uptake data were fit to both a one- and two-site model (Prism 4.0, GraphPad Software) and each curve was analyzed for accuracy of fit to the data set by the F test (P < 0.05). Data are represented as mean ± S.E.M. from replicate independent experiments conducted in duplicate. Differences were assessed by paired or unpaired Student's t test, as appropriate, with P < 0.05 considered significant.
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Results |
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11 Structure. The predicted membrane topology of mENT111 is shown in Fig. 1. The amino acid sequence corresponds to the variant of mENT1 with an arginine at position 254 (mENT1a; Genbank accession no. AF131212.1). Examination of the mENT1 gene indicates that mENT111 arises from the splicing of the 3' end of exon 10 to the 5' splice site of exon 12. This results in a frame-shift at the splice point leading to a premature stop codon after the translation of five unique C-terminal amino acids (tryptophan, glutamate, glutamine, threonine, and serine). mENT111 is predicted to have nine TM domains and cytoplasmic C and N termini.
To confirm that ENT111 did indeed encode a truncated protein. Membranes prepared from U2-OS cells transiently transfected with either p3xFLAG-mENT1, p3xFLAG-mENT111, or the empty p3xFLAG-vector were subjected to electrophoresis and immunoblotting with polyclonal rabbit anti-FLAG antibody. Bands corresponding to the predicted 33-kDa p3xFLAG-mENT111 and the 48 kDa p3xFLAG-mENT1 were detected, indicating that these proteins were expressed and were of the expected size (Fig. 2).
Tissue Distribution. RT-PCR was performed using cDNA prepared from mRNA isolated from several mouse tissues. mENT111 transcript was detected as a 1250-bp band compared with full-length mENT1 (1500 bp) on 2% agarose gels stained with ethidium bromide. These studies showed that mENT111 expression is ubiquitous, similar to the distribution of mENT1, appearing in brain, heart, kidney, liver, lung, pancreas, skeletal muscle, spleen, thymus, and testes. However, there were distinct differences in the relative ratio of mENT1 to mENT111 in some tissues (Fig. 3). Tissues with a higher relative ratio of mENT111 to mENT1 included thymus, brain, skeletal muscle and pancreas. The 1250-bp bands from skeletal muscle and brain were extracted and sequenced to confirm their identity as mENT111.
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11 in any of the human tissues examined (data not shown).
To confirm that the mENT111 protein was actually being expressed in mouse tissues, mENT1 antibody reactivity was assessed in mouse brain, testis, liver, and pancreas. Brain and testis immunoblots had bands at 45 to 50 kDa and 35 to 40 kDa, which correspond to the expected sizes of the full-length mENT1 and mENT111, respectively. However, liver had predominantly the 50-kDa mENT1, and pancreas showed predominantly the 40-kDa mENT111. The double band in liver at the 50-kDa range probably represents multiple glycosylation states of mENT1 (Vickers et al., 1999).
[3H]NBMPR Binding. PK15-mENT1 and PK15-mENT111 cells bound [3H]NBMPR with similar affinity (Kd < 0.2 nM) to more than 500,000 sites per cell (Table 2; Fig. 4A). The PK15-3xFLAG-mENT1 cells also bound [3H]-NBMPR to a single class of site with a Kd of 0.15 ± 0.01 to a maximum of 52 ± 11 x 104 sites per cell (Fig. 4B). However, [3H]NBMPR binding to the PK15-3xFLAG-mENT111 cells fit best (2 independent cell clones tested) to a two site model with 55 ± 15 x 104 sites/cell having an affinity of 1.9 ± 0.8 nM, and an additional 13.5 ± 4 x 104 sites/cell having an affinity of 0.021 ± 0.005 nM (Fig. 4B; Table 2). The untransfected PK15-NTD cell line showed no apparent specific binding of [3H]NBMPR (data not shown).
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Photolabeling of PK15-mENT1 cell membranes with [3H]-NBMPR led to a distinct radiolabeled band on SDS-PAGE gels in the 48-kDa range, which is the expected size of the mENT1 protein (Fig. 5). There was also an additional peak seen at approximately 100 kDa, possibly representing mENT1 dimers. PK15-mENT111 cell membranes were treated with [3H]NBMPR and exposed to UV light in parallel with the PK15-mENT1 membranes. The expected size of mENT111 protein was 33 kDa; however, no distinct radiolabeled peaks were observed on gels of the PK15-mENT111 proteins after exposure to [3H]NBMPR (Fig. 5), even though these same isolated membranes bound [3H]NBMPR with high affinity in reversible binding assays (data not shown).
[3H]Substrate Uptake. PK15-mENT111 cells accumulated 10 µM[3H]2-chloroadenosine (a purine substrate for ENT1) with an initial rate of transporter-mediated uptake of 0.49 ± 0.10 pmol/µl/s, to a maximum intracellular concentration of 23 ± 3 µM (Fig. 6A). This rate of uptake was similar to that discerned previously for mENT1a-transfected PK15 cells (Bone et al., 2007). To determine whether there was a change in permeant selectivity in the mENT111 variant, the uptake of the pyrimidine substrate uridine was also examined. PK15-mENT1 and PK15-mENT111 cells accumulated 100 µM[3H]uridine in a transporter-dependent manner with initial rates of 2.1 ± 0.3 and 4.4 ± 0.7 pmol/µl/s, respectively (Fig. 6B). Based on these initial time course studies, an incubation time of 15 sec was selected for the assessment of the rate of uptake of a range of concentrations of [3H]2-chloroadenosine. PK15-mENT111 cells transported 2-chloroadenosine with affinity (Km = 66 µM) similar to that of the PK15-mENT1 cells (Km = 43 µM). Likewise, there was no significant difference in the Vmax of [3H]2-chloroadenosine transport in the two cell lines (3.5 ± 1.1 and 4.4 ± 1.7pmol/µl/s for the PK15-mENT111 and PK15-mENT1a cells, respectively). (Fig. 6C). Similar studies using a 5-s incubation period conducted with the PK15-3xFLAG-mENT1 and PK15-3xFLAG-mENT111 cells revealed Km values of 44 ± 18 and 43 ± 11 µM, respectively, and Vmax values of 17 ± 4 and 19 ± 3 pmol/µl/s, respectively.
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11 cells (Fig. 7). The pseudo-Hill coefficients for all inhibitors were not significantly different from -1; therefore, the Cheng-Prusoff equation (Cheng and Prusoff, 1973) was used to calculate KI values. NBMPR was the most effective inhibitor with KI values of 0.5 ± 0.2 and 0.9 ± 0.3 nM for PK15-mENT1 and PK15-mENT111, respectively, followed by draflazine, dilazep, and dipyridamole. Adenosine was approximately 50 times more effective at inhibiting [3H]2-chloroadenosine uptake than was uridine in both the PK15-mENT1 (4 ± 2 and 200 ± 29 µM, respectively) and the PK15-mENT111 (3 ± 1 and 165 ± 71 µM, respectively). There were no significant differences in KI values (Student's paired t test, P < 0.05) for PK15-mENT111 compared with PK15-mENT1 for any of the inhibitors or substrates tested (Table 2).
Effect of NEM. Treatment of PK15-3xFLAG-mENT1 with 300 µM NEM for 30 min on ice increased both the Kd and Bmax of [3H]NBMPR binding to 3xFLAG-mENT1 compared with untreated control cells. In contrast, NEM affected only the higher affinity component of the biphasic [3H]NBMPR binding to the PK15-3xFLAG-mENT111 cells. NEM also increased both the Km and Vmax of [3H]2-chloroadenosine uptake by the full-length mENT1 but had no significant effect on the mENT111 variant (Fig. 8, Table 3). Similar uptake data (±NEM) were obtained using PK15-NTD cells transfected with either the 3xFLAG-tagged or non-FLAG-tagged versions of mENT1 and mENT111 (data not shown).
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11 in terms of substrate or inhibitor affinity. mENT1 and mENT111 are both widely expressed but differ in their relative levels of expression in a number of tissues, suggesting that the expression of the truncated variant is regulated differently than that of the full-length ENT1. Of the tissues examined, brain, thymus, skeletal muscle, and pancreas have more mENT111 transcript compared with the full-length mENT1. In contrast, liver shows mostly the full-length mENT1 transcript. A compatible profile of mENT1 immunoreactivity was also seen in these tissues; brain and testis had both a 45- to 50-kDa band and a 35- to 40-kDa band, but liver showed only the 45- to 50-kDa band, and pancreas only the 35- to 40-kDa variant. The fact that the mRNA profile does not exactly match the protein ratio of mENT1: mENT111 in some tissues (e.g., testis) may be due to differences in mRNA translation rates or stability of the two transcripts or may reflect an age related difference in the expression of the mENT1 variants (the Western blots and PCR experiments were done on tissues from different animals). The slight differences in size of the mENT1 bands between tissues are probably due to differences in the extent of glycosylation. In general, these molecular masses are a bit lower than the predicted sizes of mENT1 (50 kDa) and mENT111 (40 kDa) but are similar to those noted in other studies for photoaffinitylabeled mENT1 (Hammond and Johnstone, 1989). The tissues with high levels of mENT111 all tend to be highly metabolically active tissues, and thus one could speculate that the mENT111 variant may be expressed in response to an enhanced requirement for nucleoside salvage to support the increased metabolic activity. Unfortunately, there is insufficient functional data available in the literature at this time to confirm that these tissues actually do have enhanced nucleoside transport capacity; most studies on nucleoside transport are conducted in isolated cell lines.
The only functional differences noted between mENT1 and mENT111 were that 1) mENT111 cannot bind [3H]NBMPR irreversibly upon exposure to UV light and 2) [3H]2-chloroadenosine uptake by mENT111 is insensitive to the sulfhydryl reagent NEM. The inability of [3H]NBMPR to photolabel mENT111 suggests that the loss of this C-terminal region of mENT1 removes the residue that NBMPR normally cross-links to or that a critical residue has shifted position as a result of the structural rearrangement of the protein that is likely to occur in the truncated variant. Given that mENT111 retains the ability to bind [3H]NBMPR reversibly with high affinity, and the previous finding that the site of UV light induced covalent attachment of [3H]NBMPR to hENT1 is in the N-terminal half of the protein (Kwong et al., 1993), we would argue that the loss of the last three transmembrane domains of mENT1 leads to a conformation change that prevents covalent attachment of the [3H]NBMPR to elements of its binding pocket in the N-terminal part of the protein. It is generally believed that the S-nitrobenzyl group of NBMPR is photoactivated upon exposure to UV light; hence the amino acid residue involved in the photoaffinity labeling is probably proximal to the mENT1 region that binds the S-nitrobenzyl moiety of NBMPR (Paterson and Oliver, 1971; Young et al., 1983; Shi et al., 1984; Zhu et al., 2003).
We noted biphasic saturation profiles for [3H]NBMPR binding in some of the models tested in this study. In other cases, although not obviously biphasic, [3H]NBMPR binding had Hill coefficients of less than unity. These results probably reflect two populations of [3H]NBMPR binding proteins in these heterologous expression models, possibly representing those present at the cell surface (high affinity) and others in intracellular compartments (lower affinity). Similar biphasic [3H]NBMPR binding profiles have been described in Ehrlich ascites tumor cells (Vyas et al., 2002), endothelial cells (Hammond and Archer, 2004), and BeWo cells (Boumah et al., 1992).
Treatment of PK15-mENT1 cells with NEM increased the high-affinity binding of NBMPR and enhanced the uptake of 2-chloroadenosine by mENT. In contrast, NEM had no affect on these parameters in the PK15-mENT111 cells. There are four cysteine residues in theTM9 -C-terminal region of mENT1. These cysteines are clearly not critical for transporter function because their loss from mENT111 did not affect 2-chloroadenosine or uridine uptake or [3H]NBMPR binding. These results are compatible with a previous study on the effect of NEM on NBMPR binding to mouse Ehrlich ascites tumor cells, where we found that 100 µM NEM enhanced the ability of the nucleoside substrates uridine, adenosine, and deoxyadenosine to inhibit NBMPR binding (Vyas et al., 2002). Because treatment of the cells with NEM was conducted on ice, the NEM-induced increase in ENT1 activity was probably not due to trafficking of ENT1 protein from intracellular compartments to the plasma membrane. Rather, NEM treatment may be leading to the activation of cryptic transporters that already exist in the plasma membrane. A similar increase in ENT1-mediated nucleoside uptake, in the absence of increased plasma membrane ENT1 protein, was reported recently by Coe et al. (2002) in response to protein kinase C activation. Regardless of mechanism, this enhancement by NEM is lost in the mENT111 variant, suggesting that one or more of the four cysteines in the C-terminal region of mENT1 are involved in this effect. Thus we propose that the TM9 to C terminus region of the transporter is functionally linked to, but not directly part of, the [3H]NBMPR binding site and substrate translocation mechanism.
Little is known about the tertiary structure of ENT1. A number of studies have implicated the TMs 3-6 region in the binding of NBMPR and the translocation of substrates (Baldwin et al., 2004). Regions outside of TMs 3-6 have also been shown to affect ligand binding to ENTs, but almost all of the studies implicating specific regions/residues in ligand binding and transporter function have focused on amino acids upstream of TM9 (Visser et al., 2005b, 2007). The exceptions are a study showing that Arg404 of the Leishmania donovani nucleoside transporter LdNT1.1 (corresponding to Arg369 in TM9 of hENT1) is important for function and substrate specificity (Valdés et al., 2006) and a study showing that Leu442 in TM11 of hENT1 is important for substrate selectivity (Visser et al., 2005a). These results are difficult to reconcile with fact that mENT111, which is missing TMs 9 -11 transports both the purine nucleoside 2-chloroadenosine and the pyrimidine nucleoside uridine as effectively as the full-length mENT1. It is possible that a more detailed analysis of the substrate specificity of mENT111 will reveal subtle differences relative to mENT1. It is noteworthy that mutation of Phe334 in TM8 of hENT1 to tyrosine dramatically enhanced the turnover rate (molecules per second) of the ENT1 transporter (Visser et al., 2007), reminiscent of the effect of NEM on 2-chloroadenosine uptake seen in the present study. These results suggest that TM8 is conformationally linked to the substrate translocation mechanism in TMs 4 and 5, and cysteine residues in the region of TMs 9 -11 may affect this interaction.
The ability of ENT proteins to function despite a major C-terminal deletion is not unique to the mouse ENT1. An ENT2 splice variant lacking the last four TM domains has been identified in rabbit (rbENT2a) (Wu et al., 2005). rbENT2a was shown to be expressed to the plasma membrane and remained functional, with some slight differences in substrate affinity and inhibitor sensitivities.
In summary, we have shown that truncation of the mENT1 protein in the intracellular loop before TM9 does not affect the expression or function of the transporter upon stable transfection in PK15-NTD cells. The inability of the truncated mENT111 variant to be covalently labeled with [3H]NBMPR and its reduced sensitivity to the sulfhydryl reagent NEM highlight potential interactions between the TM 9 -11 C-terminal end of the protein and the TM 3-6 region implicated in substrate and inhibitor binding. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.
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A preliminary report of this work was presented at the XVth World Congress of Pharmacology; 2006 July 2-7; Beijing, China.
ABBREVIATIONS: ENT, equilibrative nucleoside transporter; NBMPR, nitrobenzylmercaptopurine riboside; TM, transmembrane; CK2, casein kinase II; bp, base pair(s); RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; NEM, N-ethylmaleimide; NBTGR, nitrobenzylthioguanosine riboside; CMV, cytomegalovirus; BGS, bovine growth serum; PVDF, polyvinylidene difluoride; TBS-T, Tris-buffered saline/Tween 20; NMG, N-methyl-glucamine; PAGE, polyacrylamide gel electrophoresis; PK15-NTD, pig kidney epithelial cells 15-nucleoside transport deficient; U2-OS, U2-osteosarcoma.
1 Current affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada. 
Address correspondence to: Dr. James R Hammond, Dept. of Physiology and Pharmacology, M266 Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada. E-mail: jhammo{at}uwo.ca
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) and PK15-mENT1
) cells were incubated with 10 µM[3H]2-chloroadenosine for 15 s in the presence and absence of the indicated concentrations of test agents. Each point represents the mean ± S.E.M. from at least four independent experiments conducted in duplicate.
) pretreatment with 300 µM NEM were incubated with a range of concentrations of [3H]NBMPR in the presence and absence of 10 µM NBTGR to determine the amount of specific binding to ENT1. Results are represented as Scatchard plots (bound versus bound/free), each point being the mean of five independent experiments. C and D, cells, with (
) and without (