Department of Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, California 94143-0446
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ABSTRACT |
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Nucleoside transporters are important in the disposition of nucleosides and nucleoside analogs in the kidney. Two human equilibrative nucleoside transporters have been cloned and characterized, hENT1 and hENT2. The primary goal of this study was to localize these transporters in polarized renal epithelia. hENT1 and hENT2 were tagged with green fluorescence protein, stably expressed in renal epithelial cells, and localized by immunofluorescence and functional analysis. Our data demonstrated that both transporters are expressed on the basolateral membrane. hENT1 is also present on the apical membrane. Additionally, we examined the importance to basolateral targeting of two COOH-terminal targeting motifs: a RXXV motif for hENT1 and a dileucine repeat for hENT2. Neither motif appeared to affect targeting, but the dileucine repeat was implicated in surface expression of hENT2. In addition, a splice variant of hENT2 was identified that is predicted to result in a 156-residue COOH-terminal truncation. This variant had a tissue distribution similar to wild-type hENT2 but was retained intracellularly. These data suggest that hENT1 and hENT2 on the basolateral membrane function with concentrative nucleoside transporters on the apical membrane to mediate active reabsorption of nucleosides within the kidney.
equilibrative nucleoside transporter; Madin-Darby canine kidney; dileucine; splice variant
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INTRODUCTION |
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NUCLEOSIDE TRANSPORTERS ARE polytopic membrane proteins that mediate both the uptake and release of hydrophilic nucleosides across lipophilic membranes. Nucleoside transporters are essential for cellular uptake of many clinically relevant nucleoside analogs used in the treatment of cancers and viral infections. Additionally, they are highly abundant in the kidney where they are hypothesized to play a major role in the salvage of endogenous nucleosides used for nucleotide synthesis.
Two major classes of nucleoside transporters, equilibrative nucleoside transporters (ENT, SLC29) and concentrative nucleoside transporters (CNT, SLC28), have been characterized from a variety of species, including humans and rats (12, 13, 33, 34, 40, 47, 48). The CNT family are secondary active transporters that couple cellular transport of nucleosides to an internally directed sodium or proton gradient (10, 33, 47). In contrast, the ENT family mediates passive transport of nucleosides. Classically, the ENT family can be further subdivided into two types of transporters (es and ei) based on their sensitivity to inhibition by nitrobenzylthioinosine (NBMPR); Es-type transport is sensitive to NBMPR, whereas ei-type transport is not (2, 48). Two members of the ENT family have been cloned and functionally characterized: ENT1, which mediates es-type transport, and ENT2, which mediates ei-type transport (15, 19, 48).
Members of both the CNT and ENT family are present in renal epithelium that forms the barrier between the tubule lumen and the circulatory system (11, 24, 25, 45, 46). These transporters are hypothesized to act in series to mediate the vectorial flux of nucleosides through this epithelium in a reabsorptive direction, providing a means to salvage nucleosides from the filtrate. The ability of the epithelial cells to perform this function depends on the asymmetric cellular distribution of nucleoside transporters to the apical and basolateral membrane. Early studies using apical and basolateral membrane vesicles from renal epithelium in animal models indicate that transport at the apical membrane is predominantly concentrative while basolateral transport is predominantly equilibrative (3, 23, 25, 31, 37, 39, 46). Some studies additionally report equilibrative nucleoside transport activity on the apical membrane (6, 9). Molecular localization studies in our laboratory provided the first direct evidence that CNT1 and CNT2 are localized to the apical membrane in cultured renal cells (27). This result is supported by recent immunohistochemical studies demonstrating apical expression of CNT1 in epithelia using rat kidney tissue (14). Recent work by Lai et al. (22) localized ENT1 predominantly to the basolateral membrane of differentiated renal epithelial cells. Immunofluorescence studies visualized ENT1 entirely on the basolateral membrane, but functional assays indicated low levels of ENT1-mediated transport on the apical membrane as well (22). To date, there is no information regarding intracellular localization of ENT2 in renal epithelial cells. Knowledge of the localization of these transporters will enhance our understanding of how ENT1 and ENT2 work in concert with the CNT family to mediate transepithelial flux of nucleosides and nucleoside analogs within the kidney. Furthermore, this information will contribute to understanding the differential functions of these two transporters.
In polarized cells such as renal epithelium, plasma membrane proteins are sorted in the trans-Golgi network and specifically sent to either the apical or basolateral membrane (1). Basolateral targeting appears to be triggered by distinct amino acid sequences (targeting motifs) within the protein itself, which interact with the cellular sorting machinery (50). Some of these targeting motifs [such as the tyrosine motif (NPXY) or dileucine repeat] are related to signals for clathrin-coated pit localization. These signals overlap with those used for endosomal recycling and endocytosis (1). Some proteins contain basolateral targeting motifs unrelated to clathrin-coated pits such as the R/HXXV motif seen in the cation-dependent mannose 6-phosphate receptor (CD-MPR; see Ref. 8). Although the exact mechanisms of action of these unrelated motifs are unknown, their structural orientation appears to allow for interaction with the basolateral sorting machinery. Apical targeting is less well understood but appears to be based on segregation of apical proteins into vesicles or rafts enriched with lipids preferentially delivered to the apical membrane. For some proteins, it appears that incorporation into these rafts is based on glycosylation motifs or glycosylphosphatidylinositol anchors (1).
The goal of this study was to determine the localization of both human (h) ENT1 and hENT2 within renal epithelial cells. We used Madin-Darby canine kidney (MDCK) cells, which have been successfully used to study in vivo intracellular localization of a variety of renal transporters (4, 28, 29, 38). Additionally, we sought to examine the importance of two targeting motifs in basolateral targeting and distribution of hENT1 and hENT2.
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MATERIALS AND METHODS |
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Materials. Cell culture media and supplements were purchased from the University of California, San Francisco Cell Culture Facility (San Francisco, CA). pEGFP-C1 was purchased from Clontech (Palo Alto, CA), and vector pOX was a gift from Andrew T. Gray (University of California, San Francisco). The EMBL MDCK II strain was a gift from Dr. Karl Matlin. Texas red-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Transwell polycarbonate cell culture filters and polycarbonate cell culture plates were purchased from Corning Costar (Corning, NY). Bradford reagent was purchased from Bio-Rad (Hercules, CA). Radiolabeled adenosine and thymidine were from Moravek Biochemicals (Brea, CA). All other chemicals were supplied by Sigma (St. Louis, MO).
Plasmid construction. hENT1, hENT2, and the splice variant hENT2A were cloned by PCR using primers flanking the open reading frame (ORF) of hENT1 and hENT2. The primers were designed based on published hENT1 and hENT2 cDNA sequences (7, 12). For hENT1, the sense primer was 5'-gggaaaaccgagaacaccatcaccatg-3'; the antisense primer was 5'-agtccttctgtccatcctttgtcacac-3'. For hENT2, the sense primer was 5'-ggcgcatccgccgcggcggccatggcg-3', and the antisense primer was 5'-gagcctggaggggccacttcagagcag-3'. hENT1 was then subcloned in frame into pEGFP-C1 vector by adding a Sal I site to the 5' end and a Sac II site to the 3' end. hENT2 and hENT2A were subcloned in frame into pEGFP-C1 vector by adding a Sal I site to the 5' end and an Apa I site to the 3' end. All plasmid constructions and DNA sequences were confirmed by enzyme digestion analyses and by automated sequencing at the Biomolecular Resource Center at the University of California, San Francisco.
Site-directed mutagenesis.
Mutations of hENT1 (R453A and RAIV) and of hENT2 (L455R and
LL)
were constructed with the QuickChange Site-directed Mutagenesis Kit
(Stratagene, La Jolla, CA) using wild-type hENT1 cDNA and hENT2 cDNA as
the templates. The sequences of these mutants were confirmed by DNA
sequencing at the Biomolecular Resource Center at the University of
California, San Francisco.
Stable transfection of MDCK. MDCK cells were grown in MEM with Earle's BSS supplement, 5% heat-inactivated FBS, 100 U/ml penicillin, and 100 U/ml streptomycin in a humidified atmosphere of 5% CO2-95% air at 37°C. Cells were transfected with 1 µg DNA and 16 µg Effectene (Qiagen, Valencia, CA). Cells were grown for 48 h and then diluted in media supplemented with 700 µg/ml G418. Clones were picked after 2 wk of growth in selection media, and positive clones were chosen by Western blot, confocal microscopy, and functional uptake of 3H-labeled nucleoside.
Confocal microscopy. Samples were prepared for confocal microscopy as described previously (27). Samples were grown for 4-7 days on permeable support and then fixed with 4-8% paraformaldehyde, permeabilized with 0.025% (wt/vol) saponin in PBS, stained with Texas red-conjugated phalloidin for visualization of actin, and mounted on slides in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Samples were analyzed using a Bio-Rad MRC-1024 laser scanning confocal microscope.
Functional uptake in MDCK. Stably transfected MDCK were grown for 5-7 days on permeable support and then assayed for membrane specific functionality, as described previously (27). Briefly, cells were treated with 0.1 µM [3H]inosine in choline buffer (in mM: 128 choline, 4.73 KCl, 1.25 CaCl2, 1.25 MgSO4, and 5 HEPES, pH 7.4) in the absence or presence of 1 mM inosine. All buffers in hENT2 experiments also contained 10 µM NBMPR to reduce background levels of endogenous es-type function. Reaction mix was applied to either the apical or basolateral membrane for 2 min and removed, and cells were washed three times in ice-cold choline buffer to terminate the reaction. Cellular uptake of [3H]inosine was measured by lysing cells and counting in a Beckman Scintillation Counter. All experiments were repeated in duplicate on three separate occasions.
Expression and transport assay in Xenopus laevis oocytes.
To study the function of wild-type and mutant hENTs and green
fluorescence protein (GFP)-tagged hENTs, DNA of these transporters was
subcloned in pOX vector by adding a Sal I site to the 5' end and an Xbal I site to the 3' end. pOX contains the 5' and 3'
untranslated regions of the Xenopus -globin gene flanking
the insert (17). hENT- and GFP-tagged hENT cRNA was
synthesized using T3 polymerase (Stratagene) following the
manufacturer's protocol. Oocytes were harvested and treated as
described previously (47). Fifty nanoliters of cRNA
(~0.4 ng/nl) or water was injected individually in defolliculated oocytes. Oocytes were incubated at 18°C for 30-40 h, and then uptake assays were performed for 40 min at 25°C in 100 µl of
transport buffer (2 mM KCl, 1 mM CaCl2, and 10 mM HEPES)
containing various concentrations of 3H-labeled nucleosides
(Moravek Biochemicals). The reaction was terminated by washing oocytes
five times in 3 ml ice-cold choline buffer. Oocytes were lysed
individually in 10% SDS, and the amount of radiolabeled nucleoside
transported in each oocyte was determined by liquid scintillation counting.
Statistics and data analysis. Groups of 8-10 cRNA-injected or water-injected oocytes were used for each experiment. Uptake values are expressed as means ± SE. For kinetic studies, uptake rates (V) determined at different substrate concentrations (S) were fit to the Michaelis-Menten equation: V = Vmax × S/(Km + S), where Vmax is the maximal uptake rate, and Km is the Michaelis-Menten constant (the substrate concentration at Vmax/2). Fits were carried out using a nonlinear least-squares regression-fitting program (Kaleidagraph, V.3.0; Abelbeck/Synergy Software, Reading, PA). Kinetic experiments were repeated several times in different batches of oocytes; data for one representative experiment are presented in this study. Statistical analysis was carried out by comparing the uptakes from tested compounds with those from controls in the same experiments using a two-tailed, two-sample equal-variance t-test. Results with the probability of P < 0.05 were considered statistically significant.
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RESULTS |
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Localization of hENT1 and hENT2 in polarized renal epithelial
cells.
To visualize hENT1 and hENT2 in the absence of protein-specific
antibodies, we tagged the NH2 terminus of hENT1 and hENT2 with GFP. Kinetic studies in Xenopus laevis oocytes
indicated that there were no significant differences in the uptake of
adenosine or thymidine between tagged and untagged transporters (Table
1), suggesting that the GFP tag does not
kinetically alter the function of these transporters.
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Basolateral targeting is independent of the COOH-terminal tail of
both hENT1 and hENT2.
We were interested in investigating the molecular determinants
responsible for polarized localization of hENT1 and hENT2. Based on
hydropathy plot analysis and topology studies, hENT1 and hENT2 are each
predicted to have eleven transmembrane domains with a four-amino-acid
COOH-terminal tail (Fig. 3A;
see Refs. 12-14). The COOH terminus of both
transporters contained a motif implicated in basolateral targeting: an
R/HXXV motif in hENT1 (RAIV) and a dileucine repeat in hENT2 (LL). No
other targeting motifs were obvious in either sequence at either
terminus. We investigated the significance of these two sequences on
polarized trafficking of the proteins via mutagenesis studies.
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Identification of an hENT2 variant.
In the process of cloning hENT2, using primers flanking the ORF of
the published hENT2 cDNA sequence (7), we found a
variant termed hENT2A (GenBank accession no. AF401235). We determined this to be a splice variant based on the genomic sequence of hENT2 (GenBank accession no. AF034102), which has 12 exons and 11 introns.
hENT2A uses a different splicing site on the 5' end of exon 9, causing
a 40-bp deletion (positions 1103-1142) in hENT2A mRNA (Fig.
5). This out-of-frame deletion introduces
a premature stop codon in the ORF, encoding a truncated variant that is
156 amino acids shorter than wild-type hENT2 and has an alternative COOH-terminal sequence (Fig. 6). RT-PCR
analysis of several tissues found that both wild-type and variant hENT2
are expressed in skeletal muscle, liver, lung, brain, kidney, heart,
pancreas, and placenta (data not shown).
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DISCUSSION |
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Past studies have attempted to localize equilibrative nucleoside transport within epithelia with conflicting results. Both the absence and presence of es-type transport activity (presumably ENT1) in brush-border membrane vesicles has been reported (5, 6, 9, 26). In contrast, ei-type activity (presumably ENT2) was reported to reside only in basolateral membrane vesicles (5). Because functional studies in isolated plasma membrane vesicles may be confounded by the presence of multiple transport activities or contamination with other membranes, data localizing transporters using functional activity are difficult to interpret.
In this study, we directly examined the localization of GFP-tagged hENT1 and hENT2 in renal epithelial cells. Our data demonstrate that hENT1 and hENT2 are present and functional on the basolateral membrane (Figs. 1 and 2). Interestingly, hENT1 appears in small amounts on the apical membrane where it is also functional. The function of hENT1 on the apical membrane in transfected MDCK was also demonstrated recently by Lai et al. (22). Previous data from this laboratory demonstrated that the concentrative nucleoside transporters, CNT1 and CNT2, are predominantly localized to the apical membrane in renal epithelial cells (27). Together, these data provide a picture of asymmetrically localized CNTs and ENTs working in concert to salvage nucleosides and nucleoside analogs from the tubular filtrate. In vivo studies showing that adenosine is reabsorbed in the kidney support this model (9).
We were additionally interested in examining the molecular determinants responsible for basolateral targeting of these two transporters. The COOH-terminal tail of hENT1 contained an R/HXXV motif (RAIV) that has been implicated in basolateral sorting of CD-MPR (8, 30, 51). Neither mutation nor truncation of this sequence affected hENT1 levels on the basolateral membrane (Fig. 3). In contrast, the COOH-terminal tail of hENT2 contained a dileucine repeat. This motif is implicated as a signal in both basolateral sorting and endosomal recycling of a large number of proteins (16). Both mutation and truncation of the dileucine affected surface expression of hENT2, but, in both cases, all protein that reached the plasma membrane remained confined to the basolateral membrane. This indicates that this motif is important for maintaining steady-state expression of hENT2 on the plasma membrane. Although this does not implicate the dileucine as a targeting motif, the repeat may be important in endosomal recycling or surface retention of hENT2.
Differential localization of hENT1 and hENT2 further substantiates the idea that these two transporters are maintained and regulated by distinct mechanisms within the cell. hENT1 is found ubiquitously throughout the body and is thought to be the major transporter involved in uptake of nucleosides for DNA synthesis. hENT1 is also implicated in terminating adenosine signals in the vicinity of adenosine receptors (32). Within the renal epithelium, the A1 adenosine receptor, which also localizes to both membranes in MDCK, is thought to be the major receptor involved in adenosine signaling (36). Conditions of chronic hypoxia selectively downregulate ENT1 function as a means to increase extracellular adenosine levels at the site of its receptor (21). Symbiosis between hENT1 and the A1 adenosine receptor may explain the presence of hENT1 on the apical membrane.
In contrast, hENT2 is expressed in far lower amounts in all tissues except skeletal muscle. It has a lower affinity for most physiological nucleosides, with the exception of inosine, an adenosine metabolite (32, 43). Recent data indicate that it also interacts with nucleoside bases, preferring the purinergic base hypoxanthine (49). For this reason, it has been proposed that hENT2 is involved in mechanisms requiring heavy adenosine metabolism, such as ATP depletion in skeletal muscle caused by strenuous exercise (7). Within the kidney, its purely basolateral localization suggests that its major role is to function in concert with CNTs in the salvage of nucleosides from the filtrate.
Confocal microscopy of MDCK expressing the GFP-tagged splice variant hENT2A indicates that the variant is not expressed on the surface of these cells (Fig. 7). Furthermore, our data demonstrated that the variant did not function when expressed in oocytes, suggesting that it is not functional or lacks expression on the plasma membrane. In addition, expression of hENT2A did not affect the function of wild-type hENT2, a phenomenon that has been demonstrated for spliced isoforms of other membrane proteins (20, 35, 41, 44). The role of hENT2A is unknown.
In summary, we report that cellular hENT2 is localized exclusively to the basolateral membrane and that hENT1 is localized primarily to the basolateral membrane in renal epithelial cells. The COOH-terminal dileucine motif in hENT2 is implicated in surface expression of this protein; however, neither the dileucine motif nor the RXXV motif in hENT1 appears to be important for basolateral targeting.
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ACKNOWLEDGEMENTS |
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This work was supported by General Medical Sciences Grant in Pharmaceutical Chemistry, Pharmacology and Toxicology nos. GM-07175 (L. M. Mangravite) and GM-42230 and by a grant from GlaxoSmithKline (G. Xiao).
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FOOTNOTES |
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* L. M. Mangravite and G. Xiao contributed equally to this work.
Address for reprint requests and other correspondence: K. M. Giacomini, Dept. of Biopharmaceutical Sciences, Univ. of California, San Francisco, San Francisco, CA 94143-0446 (E-mail: kmg{at}itsa.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 14, 2003;10.1152/ajprenal.00215.2002
Received 5 June 2002; accepted in final form 15 December 2002.
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