Characterization of mENT1 11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1 11) missing the last three transmembrane domains of the full-length mENT1. The mENT1 11 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 mENT1 11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1 11 in terms of transport function or inhibitor binding affinity. PK15-mENT1 11 transfected cells bound the ENT1 probe [H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [H]2-chloroadenosine and [H]uridine. The only significant differences between the mENT1 variants were that mENT1 11 could not be photolabeled with [H]NBMPR and that mENT1 11 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. Nucleosides and their therapeutic analogs are hydrophilic molecules that require a system to transport them from the extracellular environment into the cytoplasm of mammalian cells (Baldwin et al., 2004; 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 This study was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (to J.R.H.). K.R. gratefully acknowledges the financial support provided by the Schulich School of Medicine and Dentistry, University of Western Ontario, during the course of his graduate studies. D.B.J.B. is the recipient of an Ontario Graduate Scholarship in Science and Technology. A preliminary report of this work was presented at the XVth World Congress of Pharmacology; 2006 July 2–7; Beijing, China. 1 Current affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.041871. 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, Trisbuffered 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. 0026-895X/08/7401-264–273$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 41871/3353818 Mol Pharmacol 74:264–273, 2008 Printed in U.S.A. 264 at A PE T Jornals on July 4, 2017 m oharm .aspeurnals.org D ow nladed from 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 [H]NBMPR (Bone et al., 2007). We have now identified a third mENT1 variant that arises from the skipping of exon 11 during preRNA processing, designated mENT1 11 (GenBank accession no. EU180577). 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 mENT1 11 encoded a protein that retained high affinity for [H]NBMPR. The present study reports on the complete characterization of this novel splice variant. Materials and Methods Materials. Culture flasks were purchased from BD Biosciences (Franklin Lakes, New Jersey). Modified Eagle’s medium, sodium pyruvate, nonessential amino acids, G418 (Geneticin), Lipofectamine 2000, penicillin/streptomycin solution, trypsin/EDTA solution, and culturegrade phosphate-buffered saline (PBS) were purchased from Invitrogen (Burlington, ON, Canada). N-Ethylmaleimide (NEM), 2-chloroadenosine, dipyridamole [2,6-bis(diethanolamino)-4,8-dipiperidinopyridmido-[4,5-d]pyrimidine], NBMPR, nitrobenzylthioguanosine riboside [NBTGR; S-(4-nitrobenzyl)-6-thioguanosine], rabbit polyclonal anti-FLAG antibody, goat anti-rabbit antibody conjugated to horseradish peroxidase, p3 FLAG-CMV10 vector, protease inhibitor cocktail [4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, bestatin, leupeptin, and aprotinin], and mRNA purification kits were purchased from Sigma-Aldrich (Oakville, ON, Canada. The antiENT1 antibody [ENT1 (K-15); affinity-purified polyclonal], raised to the C-terminal end of the central intracellular loop of ENT1 (amino acids 250–300), was from Santa Cruz Biotechnology (Santa Cruz, CA). Draflazine [2-(aminocarbonyl)-4-amino-2,6-dichlorophenyl)-4-[5,5bis(4-fluorophenyl)pentyl]-1-piperazine acetamide 2HCl] was acquired from Janssen Research Foundation (Beerse, Belgium). Dilazep (N,N bis[3-(3,4,5-trimeth-oxybenzo-yloxy)propyl]-homo-piperazine) was provided by Asta Werke (Frankfurt, Germany). [H]NBMPR (5.5–20.1 Ci/mmol), [H]2-chloroadenosine (9.1 Ci/mmol), and H-labeled water (1 mCi/g) were purchased from Moravek Biochemicals (Brea, California). Oligonucleotide primers were purchased from SigmaGenosys (Oakville, ON, Canada), and all restriction enzymes were from Fermentas International Inc. (Burlington, ON, Canada). Platinum Taq polymerase, Superscript First-Strand Synthesis System for RT-PCR, Pure-link Gel-Extraction Kit, DH5 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). Fig. 1. Predicted topology of mENT1 11. The sequence is identical to mENT1 from the N-terminal to Gly356. Residues 357–361 are unique to mENT1 11, and residues 362–458 of mENT1 are missing from mENT1 11. Equilibrative Nucleoside Transporter Splice Variants 265 at A PE T Jornals on July 4, 2017 m oharm .aspeurnals.org D ow nladed from Plasmid Generation. mENT1 and mENT1 11 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). To generate plasmids encoding mENT1 or mENT1 11 with an N-terminal FLAG epitope tag (DYKYYYD), pcDNA3.1-mENT1 and pcDNA3.1-mENT1 11 were used as templates for PCR with primers containing the appropriate restriction enzyme cut sites (5 HindIIImENT1 and 3 mENT1-Kpn1; Table 1) for ligation into the p3 FLAG-CMV10 vector. PCR products were separated on 1.2% agarose gels containing ethidium bromide, cut with the appropriate restriction enzyme, and ligated into p3 FLAG-CMV10. DH5 subcloning 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 g/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-mENT1 11, p3 FLAG-CMV10mENT1, or p3 FLAG-CMV10-mENT1 11 using Lipofectamine 2000. The ratio of DNA to Lipofectamine 2000 was 1:3, using approximately 4.0 g of plasmid DNA. Transfected cells were selected based on survival in 500 g/ml G418, and individual cell colonies were isolated after limiting dilution of the surviving cells. The PK15mENT1 and PK15-mENT1 11 cell lines were maintained at 37°C in a 5% 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 g/ml of streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 300 g/ml G418. mRNA was collected from each cell clone and tested for the presence of the respective mENT1 or mENT1 11 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 p3 FLAG-mENT1 and p3 FLAG-mENT1 11 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 g/ml streptomycin. p3 FLAG-mENT1, p3 FLAG-mENT1 11, or empty p3 FLAG-vector (25 g/175-cm 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 2 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-cm 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 p3 FLAG-mENT1, p3 FLAG-mENT1 11, 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 3). Cell/membrane suspensions were then centrifuged (4°C, 3000g 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 g 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 SemiDry 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 antiFLAG 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. [H]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 M NEM, and then washed four times with NMG buffer to remove unreacted NEM before use in the