FASEB J. Integrated DNA Technologies
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.08-113902.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.08-113902v1
23/1/172    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Errasti-Murugarren, E.
Right arrow Articles by Pastor-Anglada, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Errasti-Murugarren, E.
Right arrow Articles by Pastor-Anglada, M.
(The FASEB Journal. 2009;23:172-182.)
© 2009 FASEB

A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum

Ekaitz Errasti-Murugarren1, Miriam Molina-Arcas1, Fco Javier Casado2 and Marçal Pastor-Anglada2,3

Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Institut de Biomedicina, CIBER EHD, Universitat de Barcelona, Barcelona, Spain

3 Correspondence: Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda Diagonal, 645 E-08028 Barcelona, Spain. E-mail: mpastor{at}ub.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleoside transporters are plasma membrane proteins essential for nucleoside salvage. Among them, human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) plays an essential role in this process because of its broader substrate selectivity and higher concentrative ability than the other members of the SLC28 protein family, hCNT1 and hCNT2. The aim of this study was to characterize an isoform of hCNT3, encoded by an alternatively spliced SLC28A3-related mRNA, the first identified for a CNT protein. This variant, named hCNT3ins, is the result of the insertion of 176 bp corresponding to an intron located between exons 2 and 3 of the gene. This insertion results in a shift of the reading frame, yielding a protein lacking 69 residues of the N terminus. hCNT3 and hCNT3ins mRNAs are simultaneously expressed both in normal and transformed cells and are differentially regulated by activation and differentiation. Because of the N-terminal deletion, hCNT3ins is retained in the endoplasmic reticulum, where it shows a typical hCNT3-related activity. hCNT3ins exhibits a shorter half-life than its plasma membrane counterpart, being degraded via a proteasome-dependent pathway. We suggest that this novel hCNT3 isoform would be involved in the salvage of intracellular nucleosides from the lumen of the endoplasmic reticulum to the cytoplasm.—Errasti-Murugarren, E., Molina-Arcas, M., Casado, F. J., Pastor-Anglada, M. A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum.


Key Words: alternative splicing • CNT • protein turnover


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUMAN CONCENTRATIVE NUCLEOSIDE transporter 3 (hCNT3) is a 691 amino acid (aa) plasma membrane protein responsible for the Na+-coupled transport of a broad variety of natural purine and pyrimidine nucleosides and nucleoside derivatives used in anticancer and antiviral therapies. hCNT3 is encoded by the gene SLC28A3 and has two unique and characteristic features that distinguish it from the other two SLC28 members, hCNT1 and hCNT2, encoded by the genes SLC28A1 and SLC28A2, respectively. The first characteristic is the unequivocal 2 Na+ per nucleoside stoichiometry of the substrate translocation cycle mediated by hCNT3. This feature suggests a high concentrative capacity for hCNT3 (1) . The second characteristic is its ability to accommodate protons in its ion-binding domain; that is, H+ is able to substitute for Na+ as one of the coupled cations during the substrate translocation cycle (2) . In fact, this is the only mammalian H+-coupled concentrative nucleoside transporter identified so far, even though its prokaryotic orthologs, such as NupC, do actually transport nucleosides in an H+-dependent manner (3) .

hCNT3 expression is relatively broad, and in absorptive epithelia, its apical localization is crucial to determine vectorial flux of nucleosides and nucleoside-derived drugs (4) . Moreover, the genes encoding hCNTs in general—but more important, hCNT3 in particular—show relatively low-sequence diversity, which suggests that hCNT3 could be biologically more critical than the other 2 SLC28-encoded members (5 , 6) . Based on their selectivity profiles, hCNT3 has, in principle, the ability to functionally replace both hCNT1 and hCNT2 in those tissues where these transporters are coexpressed, whereas the reverse is not true. However, hCNT3 is also a high-affinity adenosine concentrative transporter and its anatomical distribution—for instance, along the nephron—is compatible not only with a crucial role in absorption but also with purinergic regulation (4) .

Although increasing evidence is being provided relating nucleoside transport processes with anticancer therapy, most of the information available so far deals with the SLC29 gene family, encoding human equilibrative nucleoside transporter (hENT) proteins (7 , 8) . Information relating hCNT expression and therapeutic response is scarce, but interestingly, high hCNT3 expression has been linked to poor prognosis in chronic lymphocytic leukemia (CLL) patients. More surprisingly, hCNT3 protein staining through immunocytochemistry was mostly, if not exclusively, intracellular (9) . Evidence for the intracellular localization of what are thought to be, in principle, plasma membrane proteins may be the result of transporters resident in trafficking compartments, as seems to be the case for rCNT1 in transcytotic structures in the liver (10) . Alternatively, they may be present in intracellular organelles and play particular physiological roles. In fact, evidence for intracellular nucleoside-containing compartments is available in the literature, and ENT-type proteins and/or their related high-affinity ligand binding sites (i.e., NBTI) have been identified in mitochondria, lysosomes, endosomes, and the nuclear envelope (11 12 13) . Interestingly, evidence for intracellular CNT-type members has not yet been found.

In this study, we report the functional characterization of a unique intracellular CNT variant, the first identified so far, which is produced by a splice mRNA variant of the SLC28A3 gene and encodes a truncated hCNT3 transporter, hCNT3ins. hCNT3ins lacks the first 69 amino acid residues of the intracellular N-terminal tail of the protein. This variant is broadly expressed both in normal and transformed cell types, is exclusively located in the endoplasmic reticulum (ER), where it is active, and shows a higher turnover than that of its full-length counterpart.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Uridine ([5,6-3H], 39 Ci/mmol), thymidine ([5,6-3H], 85 Ci/mmol), and adenosine ([2-3H], 35 Ci/mmol) were purchased from Amersham Biosciences (Little Chalfont, UK). Cytidine ([5-3H(N)], 22,2 Ci/mmol), guanosine ([8-3H(N)], 15 Ci/mmol), and hypoxantine ([2,8-3H], 42,6 Ci/mmol) were from Moravek Biochemicals (Brea, CA, USA). Uridine, cytidine, adenosine, thymidine, guanosine, hypoxantine, and cycloheximide were obtained from Sigma-Aldrich (St. Louis, MO, USA). MG132 was purchased from Calbiochem (San Diego, CA, USA).

Cloning and plasmid construction
A 2.4 kb human CNT3 (hCNT3ins) fragment was amplified from the U937 cell line activated with phorbol-12-myristate-13-acetate (PMA) using the primers 5'-CTAAATGAAGAGCGCTTGGGACCT-3' (forward) and 5'-AGCATCTGTACTTCAGAGTTCCACTGG-3' (reverse), and cloned into pGem vector (BD Biosciences, Clontech, Palo Alto, CA, USA). The hCNT3 clone (GenBankTM accession number AF305210) was obtained from human kidney (14) . hCNT3 and hCNT3ins were then subcloned into KpnI and PstI sites of the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). Restriction sites were added using the following primers (restriction sites underlined): 5'-GGGGTACCGCATGGAGCTGAGGAG-3' and 5'-AACTGCAGGGAGAAGAGGCTGACC-3'. To generate green fluorescent protein (GFP) -tagged hCNT3 and hCNT3ins proteins, the corresponding cDNAs were isolated from the pcDNA3.1 vector by double digestion with PstI and HindIII and ligated into the digested (PstI and HindIII) pEGFP-C1 or pEGFPN1 vector (Clontech), respectively. The resulting constructs, hCNT3ins-pcDNA3.1 and hCNT3ins-pEGFP-N1, as well as hCNT3-pcDNA3.1 and hCNT3-pEGFP-C1, were used for transient transfection.

The epitope tag HA (YPYDVPDYA) was inserted at the C terminus of hCNT3 and hCNT3ins by polymerase chain reaction (PCR), based on the 2-side splicing by overlap extension method (15) . Using hCNT3-pcDNA3.1 and hCNT3ins-pcDNA3.1 plasmids as templates, AB and CD fragments were amplified using the following primers: A) 5'-CTTTTATGAATTCAGCCCTGTCCTGG-3'; B) 5'-CGCGTAGTCTGGGACGTCATACGGGTAAAATGTATTAGAGATCCCATTGCAGTTAAAGGTCGATGG-3'; C) 5'-TACCCGTATGACGTCCCAGACTACGCGTGAGGTCAGCCTCTTCTCCCTGCAGATATCCAGCACAGTGGCG-3'; D) 5'-GCCTAGGCCTCCAAAAAAGCCTCCTCACTAC-3'.

A second PCR amplification with 100 ng of the AB and DC fragments was performed using primers A and D to obtain the AD product. This fragment was double digested with EcoRI and StuI, and the purified product was ligated into the digested (EcoRI and StuI) hCNT3-pcDNA3.1 and hCNT3ins-pcDNA3.1 plasmids. The resulting constructs hCNT3-cHA-pcDNA3.1 and hCNT3ins-cHA-pcDNA3.1 were used for transient transfection.

Reverse transcriptase-PCR (RT-PCR) and real-time PCR
Total RNA was isolated from different cell lines, peripheral blood mononuclear cells (PBMCs), macrophages, placenta, and kidney lysates using the SV Total RNA Isolation System (Promega, Madison, WI, USA). One microgram of total DNase-treated RNA was used to generate cDNA, using M-MLV Reverse Transcriptase and random hexamers for reverse transcription (Life Technologies, Inc., Gaithersburg, MD, USA). PBMCs, monocytes, macrophages, immature dendritic cells (iDCs), and mature dendritic cells (mDCs) were isolated from healthy donors as described previously (16) . Placenta and kidney tissues were from healthy donors and kindly donated by Dr. Jordi Bellart [Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain] and Dr. José Ballarin (Fundació Puigvert, Barcelona, Spain), respectively. The following human cell lines were used: U937, derived from histiocytic lymphoma; Granta 519, derived from mantle cell lymphoma; JVM-2, derived from B-cell prolymphocytic leukemia; NP9 and NP29, derived from pancreas adenocarcinoma; MCF7 and T47D, derived from breast cancer; and HepG2, derived from hepatoma. All cell lines are commercially available, except NP cells, which were originally derived from a human pancreatic adenocarcioma by Dr. Gabriel Capellà [Institut Català d’Oncologia (ICO), Barcelona, Spain] (17) .

Oligonucleotides used for hCNT3 and hCNT3ins amplification were designed to bind in exons 2 and 3, and were as follows: 5'-GCACACTCAAACTGCTCCACC-3' and 5'-GGGCTCTGTGAAAGTTCAGC-3'. RT-PCR was performed using GoTaq® Green Master Mix (Promega) by denaturation for 5 min at 95°C, followed by 40 cycles of PCR amplification: 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 1 min, followed by a final extension of 15 min at 72°C.

Real-time quantitative PCR analysis of hCNT3 mRNA was performed as described previously (18) . For hCNT3ins amplification, Custom Taqman® Gene expression assays probes (Applied Biosystems, Foster City, CA, USA) were used. Absolute quantification of gene expression was performed by using hCNT3-pcDNA3.1 and hCNT3ins-pcDNA3.1 to construct standard curves based on serial dilutions of the plasmids. Standard curves were optimized in terms of correlation, slope, and efficiency and were run in duplicate simultaneously with the samples in the ABI Prism 7000 (Applied Biosystems). The standard curves allowed correlation of threshold cycle (CT) values of the samples with the mRNA copy number of each gene per microgram of total RNA.

Cell culture and transfection
HeLa and MDCK cells were maintained at 37°C/5% CO2 in Dulbecco modified Eagle medium (DMEM; BioWhittaker, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (v/v), 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. HeLa cells were transiently transfected with the plasmid constructs described above using Lipofectamine 2000 (Invitrogen) and following the manufacturer’s protocol. Nucleoside transport, confocal microscopy, and flow cytometry analysis were carried out 24 h after transfection. For microsome-derived vesicle experiments, cells were transfected using calcium phosphate, and vesicle isolation was carried out 36 h after transfection.

MDCK cells were plated on transwell plates (3402; 12 mm diameter, 0.3 µm pore; Corning Costar, Cambridge, MA, USA) and transfected using Lipofectamine 2000 as described previously (19) .

Preparation of microsome-derived vesicles
Vesicles obtained from the microsomal fraction of hCNT3ins- or mock-transfected HeLa cells were obtained as described previously (20 , 21) . Briefly, 4 x 108 HeLa cells were transfected with pcDNA3.1 or hCNT3ins-cHA-pcDNA3.1. Thirty-six hours after transfection, cells were resuspended in a hypotonic buffer and incubated for 20 min at 4°C. Cells were then resuspended in an isotonic buffer and broken with 10 strokes of a Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 min, and the supernatant obtained was further centrifuged at 12,000 g for 15 min. Finally, the supernatant was centrifuged at 100,000 g for 1 h. The microsome-derived vesicles were resuspended in the preload mediums (50 mM HEPES-Tris, pH 7.4; 0.1 mM MgSO4; and 225 mM KCl for sodium-dependent transport; or 100 mM KCl; 100 mM mannitol; and 20 mM HEPES, pH 7.4, for proton-dependent transport). A 3-fold enrichment for the microsomal marker calnexin was routinely obtained in these fractions.

Nucleoside and nucleobase transport assay
Uptake rates were measured as described previously (22) by exposing replicate cultures at room temperature to the appropriate 3H-labeled nucleoside and nucleobase (1 µM, 1 µCi/ml) in an uptake buffer (5.4 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, and 10 mM HEPES), supplemented with either sodium or choline chloride (137 mM). Initial rates of transport were determined by an incubation of 1 min, and transport was terminated by washing in an excess of chilled buffer.

Uptake studies in microsomal fraction-derived vesicles were carried out by the rapid filtration technique, as described previously (23) . Briefly, uptake was initiated by diluting the vesicle suspension 5-fold in an incubation medium containing the 3H-labeled substrate. Reactions were terminated by the addition of 1 ml of ice-cold STOP solution (225 mM NaCl, 50 mM HEPES-Tris buffer, and 0.1 mM MgSO4), followed by 2 washes each with 3 ml ice-cold STOP solution. To determine the sodium- and proton-dependent transport, the uptake of 3H-labeled uridine, cytidine, and guanosine (1 µM, 1 µCi/mL) was measured in the presence and absence of a sodium or proton gradient for 20 s (24 , 25) . For the sodium gradient, preloaded vesicles (50 mM HEPES-Tris, pH 7.4; 0.1 mM MgSO4; and 225 mM KCl) were incubated with 1 µM nucleoside either in a Na+ (50 mM HEPES-Tris, pH 7.4; 0.1 mM MgSO4; 75 mM KCl; 150 mM NaCl; and 3 µM valinomycin) or K+ (50 mM HEPES-Tris, pH 7.4; 0.1 mM MgSO4; 225 mM KCl; and 3 µM valinomycin) medium. For the proton gradient, preloaded vesicles (100 mM KCl, 100 mM mannitol, and 20 mM HEPES, pH 7.4) were incubated with 1 µM nucleoside either in the presence (100 mM KCl; 100 mM mannitol; 20 mM MES, pH 5.4; and 3 µM valinomycin) or the absence (100 mM KCl; 100 mM mannitol; 20 mM HEPES, pH 7.4; and 3 µM valinomycin) of H+.

Transwell transport experiments
MDCK cells were grown on transwell filters, transfected, and monitored for uptake rates, as described previously (4 , 14 , 19) . Filter inserts were washed 3 times in Na+ or Na+-free buffer, and then 1 µM 3H-labeled nucleoside (1 µM, 1 µCi/mL) was added to the apical side. Transport experiments were conducted with buffer (0.5 ml in the apical compartment and 0.5 ml in the basal compartment) containing either sodium or choline on both sides of the transwell filters. The transport experiments were terminated by aspirating the buffer, and filters were washed in chilled buffer. The whole filter was wiped with tissue to remove any excess buffer, removed from the plastic support, and counted on a scintillation counter. The cells on the filters were solubilized by 0.1% SDS and 100 mM NaOH.

Subcellular localization of hCNT3 and hCNT3ins proteins
To determine the subcellular localization of each isoform, confocal microscopy of GFP-fused chimeras was performed on a semiconfluent monolayer of transfected HeLa cells cultured on glass coverslips. To analyze plasma membrane localization, we used the fluorescent lectin WGA-TRITC (Molecular Probes, Carlsbad, CA, USA). Glass coverslip-grown cells were incubated with 1 µg/ml WGA-TRITC for 30 min at 4°C, rinsed 3 times in phosphate-buffered saline-Ca2+-Mg2+, fixed for 15 min in 3% paraformaldehyde/0.06 M sucrose, rinsed 3 times in phosphate-buffered saline, and then mounted with aqua-poly/mount coverslipping medium (Polysciences, Inc. Warrington, PA, USA). ER retention was analyzed by cotransfecting GFP-fused transporter chimeras with pDsRed2-ER (0.1 µg/cm2; BD Living Colors; Becton Dickinson, Franklin Lakes, NJ, USA), containing the signal sequence of calreticulin, a specific marker of the ER, and a C-terminal KDEL ER retention sequence. Images were obtained using an Olympus Fluoview 500 laser-scanning confocal microscope equipped with He-Ne and Ar lasers as the light source (Olympus America, Melville, NY, USA). Transfection efficiency and fluorescence intensity were determined by flow cytometry using a Cytomics FC 500 MPL Flow Cytometry System (Beckman Coulter, Fullerton, CA, USA).

To analyze the polarized membrane localization of both hCNT3 transporters, 1.7 x 105/cm2 MDCK cells were grown on glass coverslips for 24 h and then transfected as previously outlined. Cells were fixed and mounted, and images were obtained as described above. Actin was stained using 0.5 µg/µl of phalloidin-tetramethylrhodamine isothiocyanate (TRITC; Sigma-Aldrich). Actin is localized immediately closed to the plasma membrane in polarized cells grown on transwells.

In all cases, as fluorescence intensity for the hCNT3 variant in subcellular localization images was lower than that for the wild-type counterpart, the laser source intensity was increased to obtain similar fluorescence intensities to hCNT3.

Western blot analysis
Because of the lack of a suitable antibody to detect hCNT3 and hCNT3ins, we inserted the epitope tag HA at the C terminus of hCNT3 and hCNT3ins and detected the expression of these proteins using an anti-HA antibody. HeLa cells were transfected with HA-tagged hCNT3 and hCNT3ins, and 24 h after transfection, they were treated with 10 µg/ml cycloheximide and/or 20 µM MG-132, as described previously (26) . Cells were harvested at different time points after treatment and lysed using an extraction buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P40; 5 mM sodium pyrophosphate; 50 mM NaF; 1 mM sodium orthovanadate; and a protease inhibitor cocktail; complete MINI; Roche, Basel, Switzerland). Ten micrograms (for hCNT3 tranfected cells) and 60 µg (for hCNT3ins) of each protein extract was separated on 10% to 12% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Following incubation with anti-HA High Affinity Antibody (diluted 1:2000; Roche), proteins were detected using a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence detection kit (Amersham). Densitometric analysis was performed using Quantity One software (Bio-Rad, Hercules, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A novel alternative splice variant of the hCNT3 gene
hCNT3 cDNA was amplified from PMA-activated U937 cells (Fig. 1A ). When the amplified fragment was sequenced, an unexpected insertion of 176 bp at position 157 (position relative to the hCNT3 ATG start site) was identified. When hCNT3 cDNA was amplified from a kidney sample, the expected 2210-bp product was obtained, together with a 2386-bp fragment, corresponding to the transcript with the 176-bp insertion (Fig. 1A ), that we named hCNT3ins. Sequence alignment of hCNT3 and hCNT3ins showed that the 176-bp insertion is located between exons 2 and 3 (Fig. 1B ). Analysis of the SLC28A3 gene sequence showed that 20 introns are well supported, following the consensual [gt-ag] rule. Sequence analysis of the hCNT3-encoding gene, using the AceView database, suggested that this 176-bp intron could also be considered as an alternative exon. Therefore, the cloned 2386-bp fragment (hCNT3ins) is likely to correspond to an alternative splice variant of hCNT3 that incorporates a 176-bp exon between exons 2 and 3. This insertion alters the mRNA reading frame, and the resulting protein is translated using a new starting site showing an adequate Kozak consensus sequence. The predicted amino acid sequence of hCNT3ins is identical to hCNT3 but lacks the first 69 aa of the cytosolic amino-terminal domain (Fig. 1C ). Accordingly, as expected, Western blot analysis showed that the HA-tagged hCNT3ins variant (622 aa, ~60 kDa) was smaller than the HA-tagged hCNT3 protein (691 aa, ~75 kDa) (Fig. 1D ). Sequence analysis showed that the use of the original start codon for the hCNT3-encoding gene, located upstream of the insertion, may produce a short protein of 174 residues, apparently not related to any known protein that will deserve further analysis.


Figure 1
View larger version (41K):
[in this window]
[in a new window]

  		Figure 1. Identification of the hCNT3ins variant. A) RT-PCR amplification of full-length hCNT3 mRNA in PMA-activated U937 cells and kidney. The 2210-bp cDNA corresponds to hCNT3. The 2386-bp cDNA was identified as a newly found splice variant of hCNT3. B) Comparison of the sequences of hCNT3 and hCNT3ins. Bold residues indicate the 176-bp insertion and the ATG start codon for hCNT3 and hCNT3ins. Exons are indicated. C) Schematic representation of hCNT3 and hCNT3ins coding region. Translational start codon and stop codon are shown. D) Expression of hCNT3 isoforms. Western blot analysis of HeLa cells transfected with HA-tagged hCNT3 (10 µg loaded) and hCNT3ins (60 µg).

hCNT3ins is ubiquitously expressed in a variety of tissues
To analyze the expression pattern of hCNT3ins, RT-PCR was carried out in various cell lines and tissues using specific primers that allowed the detection of both hCNT3- and hCNT3ins-related mRNA (Fig. 2A ). In all samples analyzed, we detected the 446-bp product, corresponding to hCNT3, together with a 176-bp longer fragment which corresponded to hCNT3ins. The identities of these amplicons were confirmed by sequencing.


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Figure 2. Expression of hCNT3 and hCNT3ins mRNA. A) Qualitative PCR analysis of hCNT3 and hCNT3ins in different tissues and cell lines. The 446-bp PCR product corresponds to hCNT3 and the 622-bp product to hCNT3ins. B) Absolute quantitative real-time PCR of hCNT3 and hCNT3ins. Data show the number of hCNT3 and hCNT3ins mRNA copies per µg total RNA in different tissues and cell lines. The origin of the different tissues and cell lines used are indicated in the text (see Materials and Methods).

Although results indicated that the hCNT3ins variant is widely expressed in various tissues, RT-PCR did not allow the quantification of the expression of the two isoforms. For this reason, real-time PCR analysis was performed to quantify hCNT3 and hCNT3ins variants in various cell lines and tissues (Fig. 2B ). To specifically detect hCNT3 and hCNT3ins, probes annealing to exon2/exon3 and exon2b/exon3 junctions, respectively, were used. Expression of both isoforms was observed in all the samples examined. Monocytes, macrophages, dendritic cells (iDC, mDC), and the monocyte-derived cell line (U937) showed a remarkably high expression of hCNT3 and hCNT3ins. The relative quantity of each hCNT3 isoform-related mRNA differed among samples, in most cases hCNT3-related mRNA levels being higher than those associated with hCNT3ins. The hCNT3/hCNT3ins mRNA ratio ranged from 0.43 (U937+PMA) to 13.65 (kidney).

Functional expression of hCNT3 and hCNT3ins
Functional characterization of hCNT3 and hCNT3ins proteins was carried out by transient transfection of the corresponding engineered cDNA-containing vectors into HeLa and MDCK cells. We first addressed the question of whether this amino-terminal deletion could affect the hCNT3-related function. To do so, we expressed in HeLa cells the hCNT3ins variant as well as the wild-type counterpart (hCNT3) corresponding to the published sequence with GenBankTM accession number AF305210. Figure 3 shows the observed uptake rates 24 h after transfection, measured either in the presence or in the absence of sodium, of a panel of natural nucleosides (uridine, cytidine, thymidine, guanosine, and adenosine) and a nucleobase (hypoxanthine). Expression of the wild-type transporter resulted in a sodium-dependent nucleoside transport activity, with broad selectivity similar to that previously described and consistent with a hCNT3 type transporter (1 , 14 , 27) , whereas the hCNT3ins-expressing HeLa cells did not show any sodium-dependent nucleoside transport activity.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		Figure 3. Uptake of 3H-labeled nucleosides and [3H]-hypoxanthine by recombinant nucleoside transporters expressed in HeLa cells. Uptake of natural nucleosides and hypoxanthine (1 µM, 1 min) by hCNT3 (A) or hCNT3ins (B) was measured in transport medium containing 137 mM NaCl (open bars) or 137 mM choline chloride (closed bars). Sodium-dependent uptake (gray bars) is determined as the transport rate in the presence of Na+ minus the rate in its absence (choline medium). Data are expressed as means ± SE of 3 experiments carried out on different days on different batches of cells.

To test whether this novel variant may modulate hCNT3 function, cotransfection of hCNT3-encoding cDNA with increasing amounts of its corresponding hCNT3ins counterpart was carried out. hCNT3-mediated uridine uptake was unaffected by cotransfection with the splice variant, suggesting a lack of structural interaction between the isoforms (data not shown).

Subcellular localization of hCNT3ins
Confocal microscopy analysis of a GFP-fused chimera, transiently transfected into HeLa cells, was used to visualize the subcellular localization of hCNT3ins. This variant showed a different pattern of staining apparently associated with the ER (Fig. 4 ). In contrast, wild-type hCNT3 was located mainly at the plasma membrane, thus indicating that N-terminal attachment of GFP to hCNT3 does not alter its normal subcellular localization.


Figure 4
View larger version (54K):
[in this window]
[in a new window]

  		Figure 4. Subcellular localization of GFP-fused chimeras. Transient transfection of HeLa cells with pEGFPC1-fused hCNT3 (top panel) or pEGFPN1-hCNT3ins (bottom panel) revealed different subcellular localization of each isoform. hCNT3 is expressed at the plasma membrane, overlapping with WGA-TRITC staining (A, top panel), but not with that of pDsRed2-ER (B, top panel), a protein expressed in the endoplasmic reticulum. However, hCNT3ins does not colocalize with the plasma membrane marker WGA-TRITC (A, bottom panel) but overlaps with pDsRed2-ER staining (B, bottom panel), indicating that this variant is retained in the endoplasmic reticulum. In the subcellular localization images of the hCNT3 variant, the laser source intensity was increased to obtain similar fluorescence intensities to hCNT3.

Membrane insertion was further analyzed by colocalization analysis of both isoforms with the plasma membrane marker WGA-TRITC (Fig. 4A ). The data confirmed the cell surface expression of the wild-type transporter and the inability of the hCNT3ins isoform to traffic into the plasma membrane. The intracellular localization of each isoform was determined by cotransfection of wild-type hCNT3 and hCNT3ins with pDsRed2-ER, containing the signal sequence of calreticulin, a specific marker of the ER, and a C-terminal KDEL ER retention sequence. As shown in Fig. 4B , wild-type hCNT3 did not colocalize with the ER marker, being mainly expressed at the cell surface, whereas the truncated transporter showed a distribution pattern coincident with the ER marker, with no other structures stained. Moreover, flow cytometry analysis showed the same transfection rates for both isoforms (data not shown) but different fluorescence intensity values, being much higher for the wild-type than for the truncated variant (201 arbitrary units vs. 15.7 arbitrary units, respectively), which indicated that the expression levels of hCNT3ins were much lower than those of hCNT3.

Turnover of hCNT3 and hCNT3ins proteins
We addressed the question of whether the apparent low levels of hCNT3ins protein were the result of a shorter half-life than that of its full-length counterpart. HeLa cells were transfected with HA-tagged hCNT3 and hCNT3ins and were treated for different times with the translation inhibitor cycloheximide (Fig. 5 ). Interestingly, 2 h after treatment, hCNT3ins expression was already decreased by 60%, whereas hCNT3 levels remained unchanged, thus indicating a shorter half-life for the truncated variant (1.5 h) than for its wild-type counterpart ({approx}8 h).


Figure 5
View larger version (58K):
[in this window]
[in a new window]

  		Figure 5. Turnover of hCNT3 and hCNT3ins. A) Western blot analysis of HA-hCNT3 and HA-hCNT3ins in total cell extracts from transfected HeLa cells, treated with cycloheximide (CHX, 10 µg/mL) either in the presence or the absence of MG132 (MG, 20 µM) for the indicated time periods. Controls were untreated cells at 0 and 6 h. A representative Western blot is shown. B) The intensity of hCNT3 and hCNT3ins protein bands after treatment was measured densitometrically and normalized to the control. Results are means ± SE of 4 independent experiments.

To further clarify the mechanisms of hCNT3ins degradation, cells were treated with the proteasome inhibitor MG132. Results showed that inhibition of the proteasome increased the expression of hCNT3ins without affecting the levels of hCNT3 (data not shown). Moreover, simultaneous treatment of cycloheximide and MG132 partially blocked the rapid degradation of hCNT3ins (Fig. 5) , without affecting the levels of hCNT3 (data not shown). Under these conditions, the expression of hCNT3ins reached similar levels to those of its wild-type counterpart.

hCNT3ins characterization in a polarized model
Because hCNT3 is an apically located transporter in epithelia and its expression is known to determine nucleoside vectorial flux (4 , 14) , we proceeded to determine, first, whether the hCNT3 variant could be normally inserted into the plasma membrane in a polarized manner and, second, whether this would affect nucleoside transport across the epithelial barrier. We found that, whereas hCNT3 is localized mostly at the apical membrane of the epithelial barrier, its truncated counterpart is also found on basal membranes (Fig. 6A ). Nevertheless, hCNT3ins-associated fluorescence was actually 5-fold lower than that observed for hCNT3 because of the low expression of the protein. hCNT3- and hCNT3ins-associated biological function was also characterized in this polarized cell model. Initial rates of uridine uptake, both at the apical and the basolateral domains, were analyzed in MDCK cells grown on transwell plates and transiently transfected with either hCNT3 or its truncated variant (Fig. 6B ). Sodium-dependent uridine transport activity was mostly apically present in hCNT3-expressing MDCK cells, whereas the hCNT3ins variant completely lost the apically preferential insertion in this cell system. However, some sodium-dependent activity could be detected at both poles of hCNT3ins-expressing cells (Fig. 6B ). Although absolute uptake rates via the hCNT3ins transporter were dramatically lower than those found at the apical side of hCNT3-expressing MDCK cells, its occurrence did allow its functional analysis. In fact, as indicated by the fluorescence intensity values and the half-life experiments, the lower transport rates exhibited by the variant were the result of its lower expression levels. As shown in Fig. 6C, D , the sodium-dependent uptake rates of a panel of natural nucleosides (uridine, cytidine, thymidine, guanosine, and adenosine) showed an apparently identical selectivity for both isoforms, thus suggesting that the hCNT3ins variant is functional and able to translocate nucleosides, consistent with a broad selectivity transporter, as is the case for hCNT3.


Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		Figure 6. Characterization of hCNT3ins in polarized MDCK cells. A) MDCK cells transiently transfected with GFP-tagged hCNT3 and hCNT3ins. Cells were fixed, stained for actin with Texas Red-conjugated phalloidin, and visualized by confocal microscopy. Red = actin; green = GFP. In the subcellular localization images of the hCNT3 variant, the laser source intensity was increased to obtain similar fluorescence intensities to hCNT3. B) Sodium-dependent uridine uptake (1 µM, 1 min) into apical (open bars) and basal (closed bars) compartments of polarized hCNT3- or hCNT3ins-transfected MDCK cells. C, D) Sodium-dependent apical uptake of 3H-labeled nucleosides by recombinant nucleoside transporters expressed in polarized MDCK cells. Uptake of nucleosides (1 µM, 1 min) by hCNT3 (C) or hCNT3ins (D) was measured in transport medium containing 137 mM NaCl or 137 mM choline chloride. Apical sodium-dependent transport was calculated as uptake in NaCl medium minus uptake in choline chloride medium. Data are expressed as means ± SE of 3 experiments carried out on different days on different batches of cells.

hCNT3ins function in the ER
Once the functionality of the variant was demonstrated, we also focused on determining whether hCNT3ins-related activity could be detected in the ER. To do so, we isolated microsomes from hCNT3ins- or mock-transfected cells, and expression of hCNT3ins protein was demonstrated by Western blot analysis (Fig. 7A ). Uridine, cytidine, and guanosine uptake was measured in microsome-derived vesicles in the presence of sodium or proton gradients (Fig. 7B ). Results showed sodium-dependent transport of all 3 nucleosides in the hCNT3ins-transfected vesicles. When vesicles were assayed in the presence of a proton gradient, hCNT3ins-mediated transport of uridine and cytidine, but not guanosine, was observed, consistent with previously described data for hCNT3 (2) . These results suggest that although hCNT3ins is located intracellularly, mostly in the ER, it is biologically active.


Figure 7
View larger version (34K):
[in this window]
[in a new window]

  		Figure 7. hCNT3ins-related activity in microsomal-derived vesicles. A) Western blot analysis of the microsomal-derived vesicles of HeLa cells transfected with pcDNA3 or HA-tagged hCNT3ins. B) Uptake of uridine, cytidine, and guanosine (1 µM) was measured in microsomal-derived vesicles from hCNT3ins- and mock-transfected cells over a 20-s period. For the sodium gradient, preloaded vesicles were incubated with 1 µM nucleoside either in a Na+ or K+ medium. Sodium-dependent uptake was calculated as the transport rate in the presence of Na+ minus the rate in its absence. For the proton gradient, preloaded vesicles were incubated with 1 µM nucleoside either in the presence or the absence of H+. Proton-dependent uptake was calculated as the difference between that in each medium. Results are shown as sodium- or proton-dependent uptake rates and are expressed as means ± SE of 4 measurements.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The occurrence of nucleoside transporters in intracellular structures has so far been demonstrated for members of the SLC29 gene family only. In particular, hENT3, encoded by the gene SLC29A3, is a lysosomal membrane transport protein that translocates uridine and adenosine, but with much lower apparent affinity than its plasma membrane-localized counterparts, hENT1 and hENT2 (12) . Because hENT3 activity is optimal at acidic pH values (5.5), it is likely that this protein may be responsible for nucleoside recycling from the lysosomal compartment. In fact, the classical idea that plasma membrane NT proteins contribute to nucleoside salvage in most cell types lacking endogenous purine and pyrimidine nucleotide de novo biosynthesis, could also be translated into single cells in which nucleosides might also be salvaged from intracellular stores for macromolecule synthesis and other purposes.

hCNT proteins, encoded by SLC28 genes, belong to a different family from those encoded by SLC29 (28 , 29) . Accordingly, they bear no homology to any hENT-type protein. Moreover, because of the well-known Na+-coupling in their substrate translocation cycle, they have been assumed to be localized only at the plasma membrane, or at least to be functional exclusively in this location. Nevertheless, CNT-type proteins have been identified in intracellular structures in hepatocytes. CNT1 was found in transcytotic structures, probably reflecting its traffic from the basal to the apical plasma membrane compartments in a highly polarized cell type such as the hepatocyte (10) . Alternatively, CNT2 might also be present in intracellular structures in liver parenchymal cells because it has been reported that bile acids can increase CNT2-related activity by a short-term mechanism, which is consistent with CNT2 recruitment from intracellular stores into the plasma membrane (30) . However, no evidence for any intracellular resident hCNT-type protein, with a presumably functional role, has been reported so far.

In this study, a novel hCNT3 variant has been identified lacking 69 amino acids from its N-terminal tail, which results in an ER resident transporter that is functional. Previous studies have demonstrated the existence of alternative splice variants of ENT transporters (31 32 33 34) ; however, this is the first evidence for a splice variant of an hCNT-type transporter. In a polarized model, this hCNT3 variant produced sodium-dependent activity with a similar selectivity to the wild-type counterpart. However, when hCNT3ins is located in the ER, it is likely to use H+ gradients, instead of Na+, as its coupled cation to drive nucleoside efflux from the ER lumen. In fact, hCNT3 is the only SLC28 member that can accept one proton in place of one of the two sodium ions known to be translocated along with each nucleoside when the transporter is functional at the plasma membrane (2) . In additions, the H+ binding site seems to be localized in the far C-terminal region of the transporter (35) , which would be consistent with the N terminus domain being less relevant in the substrate translocation process. Actually, the prokaryote ortolog of hCNTs NupC lacks the N-tail and the first 3 transmembrane domains as well and is fully functional (3) . The occurrence of 1 proton/nucleoside stoichiometry is also indicated by the description of a rare hCNT3 polymorphic variant (C602R), recently identified and functionally characterized (14) . This hCNT3C602R transporter appears to translocate nucleosides in a manner compatible with 1 cation/nucleoside stoichiometry, regardless of whether this cation is a sodium ion or a proton. The existence of proton-coupled transporters in the ER has also been reported for other nonrelated proteins (36) , and this may be functionally relevant because the possibility of transient luminal acidification in the ER has been postulated (37) and expression of H+-ATPases in the ER has also been reported (38) .

It is traditionally assumed that nucleoside plasma membrane transporters play a role in nucleoside salvage, and this appears to be crucial for nucleoside and energy homeostasis because de novo nucleotide synthesis is energetically costly. However, the occurrence of NT proteins—in this particular case, hCNT3ins—in intracellular structures would be consistent with a need of subcellular storage of nucleosides in particular compartments. In fact, there is the possibility that the ER might contribute to intracellular nucleoside recycling because active nucleoside metabolism is anticipated, considering the abundance of nucleoside-related enzymes and the occurrence of UDP sugars and other high-energy nucleotide molecules in the ER (39 40 41 42 43 44 45) .

The possibility that hCNT3ins plays a crucial role in cell physiology arises from the fact that its expression is broad and, apparently, regulated. The copy number ratio of the hCNT3- and hCNT3ins-related mRNA is variable among tissues and cell types but is also dramatically modified after treatment of U937 cells with the phorbol ester PMA. This finding is interesting and deserves further analysis. Differential regulation of alternative splice variants in several genes has been described previously. In U937 cells, PMA treatment increases the expression of PECAM and changes the pattern of the splice variants expressed (46) . Nevertheless, the hCNT3 protein—reported by others (9) to be localized intracellularly in CLL cells and suggested to be associated with disease outcome—cannot be related at all to hCNT3ins, because hCNT3 staining in CLL cells was assessed using an antibody raised against the N terminus tail, which is absent in hCNT3ins. Thus, intracellular localization of hCNT proteins might occur in transformed cells (9 , 47) , but this could be the result of altered transporter sorting into the plasma membrane rather than the existence of novel variants, such as the one reported in this study. In fact, although hCNT3ins is expressed in tumor-derived cell lines, there is no apparent difference in the hCNT3/hCNT3ins ratio among transformed and normal cell types and tissues, although this particular issue also deserves further analysis.

What is evident from our findings is that both transporter proteins are expressed at variable levels and show remarkable differences in their apparent turnover rates. hCNT3ins shows a much shorter half-life than hCNT3, and its degradation is mainly, but not totally, accounted for by the proteasome pathway. Although shorter half-life values have been reported for misfolded proteins in the ER (48) , this has also been reported for properly folded proteins (49) . The latter case would appear to apply to this novel hCNT3ins variant because it is biologically functional in the ER, broadly expressed, and differentially regulated, and there is the possibility that it might support nucleoside recycling inside cells. Evidence for functional plasma membrane transporters resident in the ER has been provided (50 51 52) , although they may correspond to mutated variants (50) . However, a role for alternative splicing in determining different subcellular localization for two variants of the same transporter protein has been described in the case of the UDP-galactose carrier (51) .

Moreover, in addition to describing a novel transporter protein whose physiological roles are as yet ill defined, this study provides a basis for further structure-function studies that are currently being performed. This variant highlights the relevance of the N-terminal tail in ER export and correct trafficking of an hCNT-type transporter into the plasma membrane. Moreover, hCNT3ins is inserted in the plasma membrane in a polarized model, although it is localized both in the apical and basal membrane, whereas hCNT3 is inserted in the apical membrane. Taken together, these results suggest a different role for the N-terminal tail in polarized and nonpolarized models. Interestingly, coexpression of hCNT3 and hCNT3ins did not result in a dominant negative effect, as described for other transporter proteins (34) , which can be interpreted either as evidence of a monomer structure of the functionally active hCNT3 transporter or as evidence of a need for the N-terminal domain for polymerization. If the latter is true, then it is also evident from our data that hCNT3ins monomers do show functional activity, even though they are expressed at very low levels.

In summary, this study provides what we believe is the first evidence for a splice variant of an hCNT-type transporter that is expressed in the ER, probably contributing to intracellular nucleoside recycling. This natural variant provides the first structure-function evidence for the N-terminal domain of the hCNT3 transporter in intracellular trafficking and appropriate insertion into the plasma membrane. The fact that it is an hCNT3-isoform-related variant that is expressed in intracellular compartments seems reasonable because hCNT3 is the only one in the SLC28 protein family that can translocate nucleosides in a proton-dependent manner. Moreover, its broad occurrence in transformed and normal cell types suggests that hCNT3ins is a major player in intracellular nucleoside metabolism, although this issue should be addressed further. We propose the possibility that splice variants resulting in intracellular retention of a plasma membrane transporter would be evolutionarily enhanced by the ability of a transporter protein to translocate either Na+ or H+, the former for its plasma membrane localization and the latter for intracellular structures—a unique feature not present in the other hCNT family members.


   ACKNOWLEDGMENTS
 
This work was supported by grants from Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo (PI-020934), Ministerio de Educación y Ciencia (BFU2006-07556/BFI), and Fundación Ramón Areces to F.J.C.; and from Ministerio de Educación y Ciencia e Innovación (SAF2005-01259 and SAF2008-00577), Fundación para la Investigación y la Prevención del SIDA en España (FIPSE), Centro de Investigación Biomédica en Red (CIBER-EHD), Ministerio de Sanidad y Consumo, and Departament d’Universitats, Recerca i Socitat de la Informació (DURSI), Generalitat de Catalunya (2005SGR00315) to M.P.-A. E.E.-M. was the recipient of a Formación de Profesorado Universitario (FPU) fellowship from Ministerio de Educación y Ciencia.


   FOOTNOTES
 
1 Co-first authors.

2 These authors contributed equally to this work.

Received for publication June 12, 2008. Accepted for publication September 4, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ritzel, M. W., Ng, A. M., Yao, S. Y., Graham, K., Loewen, S. K., Smith, K. M., Ritzel, R. G., Mowles, D. A., Carpenter, P., Chen, X. Z., Karpinski, E., Hyde, R. J., Baldwin, S. A., Cass, C. E., Young, J. D. (2001) Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J. Biol. Chem. 276,2914-2927[Abstract/Free Full Text]
  2. Smith, K. M., Slugoski, M. D., Loewen, S. K., Ng, A. M., Yao, S. Y., Chen, X. Z., Karpinski, E., Cass, C. E., Baldwin, S. A., Young, J. D. (2005) The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics. J. Biol. Chem. 280,25436-25449[Abstract/Free Full Text]
  3. Loewen, S. K., Yao, S. Y., Slugoski, M. D., Mohabir, N. N., Turner, R. J., Mackey, J. R., Weiner, J. H., Gallagher, M. P., Henderson, P. J., Baldwin, S. A., Cass, C. E., Young, J. D. (2004) Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes. Mol. Membr. Biol. 21,1-10[CrossRef][Medline]
  4. Rodriguez-Mulero, S., Errasti-Murugarren, E., Ballarin, J., Felipe, A., Doucet, A., Casado, F. J., Pastor-Anglada, M. (2005) Expression of concentrative nucleoside transporters SLC28 (CNT1, CNT2, and CNT3) along the rat nephron: effect of diabetes. Kidney Int. 68,665-672[CrossRef][Medline]
  5. Badagnani, I., Chan, W., Castro, R. A., Brett, C. M., Huang, C. C., Stryke, D., Kawamoto, M., Johns, S. J., Ferrin, T. E., Carlson, E. J., Burchard, E. G., Giacomini, K. M. (2005) Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3). Pharmacogenomics J. 5,157-165[CrossRef][Medline]
  6. Damaraju, S., Zhang, J., Visser, F., Tackaberry, T., Dufour, J., Smith, K. M., Slugoski, M., Ritzel, M. W., Baldwin, S. A., Young, J. D., Cass, C. E. (2005) Identification and functional characterization of variants in human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), arising from single nucleotide polymorphisms in coding regions of the hCNT3 gene. Pharmacogenet. Genomics 15,173-182[Medline]
  7. Molina-Arcas, M., Marce, S., Villamor, N., Huber-Ruano, I., Casado, F. J., Bellosillo, B., Montserrat, E., Gil, J., Colomer, D., Pastor-Anglada, M. (2005) Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells. Leukemia 19,64-68[Medline]
  8. Spratlin, J., Sangha, R., Glubrecht, D., Dabbagh, L., Young, J. D., Dumontet, C., Cass, C., Lai, R., Mackey, J. R. (2004) The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin. Cancer Res. 10,6956-6961[Abstract/Free Full Text]
  9. Mackey, J. R., Galmarini, C. M., Graham, K. A., Joy, A. A., Delmer, A., Dabbagh, L., Glubrecht, D., Jewell, L. D., Lai, R., Lang, T., Hanson, J., Young, J. D., Merle-Beral, H., Binet, J. L., Cass, C. E., Dumontet, C. (2005) Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabine-sensitive and -insensitive populations. Blood 105,767-774[Abstract/Free Full Text]
  10. Duflot, S., Calvo, M., Casado, F. J., Enrich, C., Pastor-Anglada, M. (2002) Concentrative nucleoside transporter (rCNT1) is targeted to the apical membrane through the hepatic transcytotic pathway. Exp. Cell Res. 281,77-85[CrossRef][Medline]
  11. Lai, Y., Tse, C. M., Unadkat, J. D. (2004) Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. J. Biol. Chem. 279,4490-4497[Abstract/Free Full Text]
  12. Baldwin, S. A., Yao, S. Y., Hyde, R. J., Ng, A. M., Foppolo, S., Barnes, K., Ritzel, M. W., Cass, C. E., Young, J. D. (2005) Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes. J. Biol. Chem. 280,15880-15887[Abstract/Free Full Text]
  13. Mani, R. S., Hammond, J. R., Marjan, J. M., Graham, K. A., Young, J. D., Baldwin, S. A., Cass, C. E. (1998) Demonstration of equilibrative nucleoside transporters (hENT1 and hENT2) in nuclear envelopes of cultured human choriocarcinoma (BeWo) cells by functional reconstitution in proteoliposomes. J. Biol. Chem. 273,30818-30825[Abstract/Free Full Text]
  14. Errasti-Murugarren, E., Cano-Soldado, P., Pastor-Anglada, M., Casado, F. J. (2008) Functional characterization of a nucleoside-derived drug transporter variant (hCNT3C602R) showing altered sodium-binding capacity. Mol. Pharmacol. 73,379-386[Abstract/Free Full Text]
  15. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77,61-68[CrossRef][Medline]
  16. Minuesa, G., Purcet, S., Erkizia, I., Molina-Arcas, M., Bofill, M., Izquierdo-Useros, N., Casado, F. J., Clotet, B., Pastor-Anglada, M., Martinez-Picado, J. (2008) Expression and functionality of anti-human immunodeficiency virus and anticancer drug uptake transporters in immune cells. J. Pharmacol. Exp. Ther. 324,558-567[Abstract/Free Full Text]
  17. Villanueva, A., Garcia, C., Paules, A. B., Vicente, M., Megias, M., Reyes, G., de Villalonga, P., Agell, N., Lluis, F., Bachs, O., Capella, G. (1998) Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells. Oncogene 17,1969-1978[CrossRef][Medline]
  18. Molina-Arcas, M., Bellosillo, B., Casado, F. J., Montserrat, E., Gil, J., Colomer, D., Pastor-Anglada, M. (2003) Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia. Blood 101,2328-2334[Abstract/Free Full Text]
  19. Harris, M. J., Kagawa, T., Dawson, P. A., Arias, I. M. (2004) Taurocholate transport by hepatic and intestinal bile acid transporters is independent of FIC1 overexpression in Madin-Darby canine kidney cells. J. Gastroenterol. Hepatol. 19,819-825[CrossRef][Medline]
  20. Leuzzi, R., Fulceri, R., Marcolongo, P., Banhegyi, G., Zammarchi, E., Stafford, K., Burchell, A., Benedetti, A. (2001) Glucose 6-phosphate transport in fibroblast microsomes from glycogen storage disease type 1b patients: evidence for multiple glucose 6-phosphate transport systems. Biochem. J. 357,557-562[CrossRef][Medline]
  21. Banhegyi, G., Marcolongo, P., Fulceri, R., Hinds, C., Burchell, A., Benedetti, A. (1997) Demonstration of a metabolically active glucose-6-phosphate pool in the lumen of liver microsomal vesicles. J. Biol. Chem. 272,13584-13590[Abstract/Free Full Text]
  22. Duflot, S., Riera, B., Fernandez-Veledo, S., Casado, V., Norman, R. I., Casado, F. J., Lluis, C., Franco, R., Pastor-Anglada, M. (2004) ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors. Mol. Cell. Biol. 24,2710-2719[Abstract/Free Full Text]
  23. Ruiz-Montasell, B., Javier Casado, F., Felipe, A., Pastor-Anglada, M. (1992) Uridine transport in basolateral plasma membrane vesicles from rat liver. J. Membr. Biol. 128,227-233[Medline]
  24. Ngo, L. Y., Patil, S. D., Unadkat, J. D. (2001) Ontogenic and longitudinal activity of Na(+)-nucleoside transporters in the human intestine. Am. J. Physiol. 280,G475-G481
  25. Hu, H., Endres, C. J., Chang, C., Umapathy, N. S., Lee, E. W., Fei, Y. J., Itagaki, S., Swaan, P. W., Ganapathy, V., Unadkat, J. D. (2006) Electrophysiological characterization and modeling of the structure activity relationship of the human concentrative nucleoside transporter 3 (hCNT3). Mol. Pharmacol. 69,1542-1553[Abstract/Free Full Text]
  26. Gorner, K., Holtorf, E., Odoy, S., Nuscher, B., Yamamoto, A., Regula, J. T., Beyer, K., Haass, C., Kahle, P. J. (2004) Differential effects of Parkinson’s disease-associated mutations on stability and folding of DJ-1. J. Biol. Chem. 279,6943-6951[Abstract/Free Full Text]
  27. Toan, S. V., To, K. K., Leung, G. P., de Souza, M. O., Ward, J. L., Tse, C. M. (2003) Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells. Plügers Arch. 447,195-204[CrossRef][Medline]
  28. Gray, J. H., Owen, R. P., Giacomini, K. M. (2004) The concentrative nucleoside transporter family, SLC28. Plügers Arch. 447,728-734[CrossRef][Medline]
  29. Baldwin, S. A., Beal, P. R., Yao, S. Y., King, A. E., Cass, C. E., Young, J. D. (2004) The equilibrative nucleoside transporter family, SLC29. Plügers Arch. 447,735-743[CrossRef][Medline]
  30. Fernandez-Veledo, S., Huber-Ruano, I., Aymerich, I., Duflot, S., Casado, F. J., Pastor-Anglada, M. (2006) Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells. Biochem. J. 395,337-344[CrossRef][Medline]
  31. Griffiths, M., Yao, S. Y., Abidi, F., Phillips, S. E., Cass, C. E., Young, J. D., Baldwin, S. A. (1997) Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem. J. 328(Pt. 3),739-743[Medline]
  32. Wu, S. K., Ann, D. K., Kim, K. J., Lee, V. H. (2005) Fine tuning of rabbit equilibrative nucleoside transporter activity by an alternatively spliced variant. J. Drug Target. 13,521-533[CrossRef][Medline]
  33. Handa, M., Choi, D. S., Caldeiro, R. M., Messing, R. O., Gordon, A. S., Diamond, I. (2001) Cloning of a novel isoform of the mouse NBMPR-sensitive equilibrative nucleoside transporter (ENT1) lacking a putative phosphorylation site. Gene 262,301-307[CrossRef][Medline]
  34. Mangravite, L. M., Xiao, G., Giacomini, K. M. (2003) Localization of human equilibrative nucleoside transporters, hENT1 and hENT2, in renal epithelial cells. Am. J. Physiol. 284,F902-F910
  35. Slugoski, M. D., Smith, K. M., Mulinta, R., Ng, A. M., Yao, S. Y., Morrison, E. L., Lee, Q. O., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., Young, J. D. (2008) A conformationally mobile cysteine residue (C561) modulates Na+- and H+-activation of human concentrative nucleoside transporter 3 (hCNT3). J. Biol. Chem. 283,24922-24934[Abstract/Free Full Text]
  36. Rubio-Aliaga, I., Boll, M., Vogt Weisenhorn, D. M., Foltz, M., Kottra, G., Daniel, H. (2004) The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1. J. Biol. Chem. 279,2754-2760[Abstract/Free Full Text]
  37. Monni, M., Roberti, R., Corazzi, L. (2001) Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum. Eur. J. Biochem. 268,2020-2027[Medline]
  38. Rees-Jones, R., Al-Awqati, Q. (1984) Proton-translocating adenosinetriphosphatase in rough and smooth microsomes from rat liver. Biochemistry 23,2236-2240[CrossRef][Medline]
  39. Wachstein, M., Fernandez, C. (1964) Electron microscopic localization of nucleoside triphosphatase in endoplasmic reticulum of liver and pancreas. J. Histochem. Cytochem. 12,40-42[Medline]
  40. Goldfischer, S., Essner, E., Schiller, B. (1971) Nucleoside diphosphatase and thiamine pyrophosphatase activities in the endoplasmic reticulum and golgi apparatus. J. Histochem. Cytochem. 19,349-360[Abstract]
  41. Bischoff, E., Tran-Thi, T. A., Decker, K. F. (1975) Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum. Eur. J. Biochem. 51,353-361[Medline]
  42. Bischoff, E., Wilkening, J., Tran-Thi, T. A., Decker, K. (1976) Differentiation of the nucleotide pyrophosphatases of rat-liver plasma membranes and endoplasmic reticulum by enzymic iodination. Eur. J. Biochem. 62,279-283[Medline]
  43. Franco, R., Canela, E. I., Bozal, J. (1986) Enzymes of the purine metabolism in rat brain microsomes. Neurochem. Res. 11,407-422[CrossRef][Medline]
  44. Failer, B. U., Braun, N., Zimmermann, H. (2002) Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase. J. Biol. Chem. 277,36978-36986[Abstract/Free Full Text]
  45. Kapetanovich, L., Baughman, C., Lee, T. H. (2005) Nm23H2 facilitates coat protein complex II assembly and endoplasmic reticulum export in mammalian cells. Mol. Biol. Cell 16,835-848[Abstract/Free Full Text]
  46. Wang, Y., Su, X., Sorenson, C. M., Sheibani, N. (2003) Modulation of PECAM-1 expression and alternative splicing during differentiation and activation of hematopoietic cells. J. Cell Biochem. 88,1012-1024[CrossRef][Medline]
  47. Gloeckner-Hofmann, K., Guillen-Gomez, E., Schmidtgen, C., Porstmann, R., Ziegler, R., Stoss, O., Casado, F. J., Ruschoff, J., Pastor-Anglada, M. (2006) Expression of the high-affinity fluoropyrimidine-preferring nucleoside transporter hCNT1 correlates with decreased disease-free survival in breast cancer. Oncology 70,238-244[CrossRef][Medline]
  48. Alfalah, M., Krahn, M. P., Wetzel, G., von Horsten, S., Wolke, C., Hooper, N., Kalinski, T., Krueger, S., Naim, H. Y., Lendeckel, U. (2006) A mutation in aminopeptidase N (CD13) isolated from a patient suffering from leukemia leads to an arrest in the endoplasmic reticulum. J. Biol. Chem. 281,11894-11900[Abstract/Free Full Text]
  49. Ayalon-Soffer, M., Kamhi-Nesher, S., Lederkremer, G. Z. (1999) Folding and self-assembly do not prevent ER retention and proteasomal degradation of asialoglycoprotein receptor H2a. FEBS Lett. 460,112-116[CrossRef][Medline]
  50. Pasyk, E. A., Foskett, J. K. (1995) Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J. Biol. Chem. 270,12347-12350[Abstract/Free Full Text]
  51. Kabuss, R., Ashikov, A., Oelmann, S., Gerardy-Schahn, R., Bakker, H. (2005) Endoplasmic reticulum retention of the large splice variant of the UDP-galactose transporter is caused by a dilysine motif. Glycobiology 15,905-911[Abstract/Free Full Text]
  52. Bidaux, G., Flourakis, M., Thebault, S., Zholos, A., Beck, B., Gkika, D., Roudbaraki, M., Bonnal, J. L., Mauroy, B., Shuba, Y., Skryma, R., Prevarskaya, N. (2007) Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function. J. Clin. Invest. 117,1647-1657[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.08-113902v1
23/1/172    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Errasti-Murugarren, E.
Right arrow Articles by Pastor-Anglada, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Errasti-Murugarren, E.
Right arrow Articles by Pastor-Anglada, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS