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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of 1Medicine, 2Molecular Pharmacology, and 3Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York; 4Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and 5Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan
Submitted 16 May 2007 ; accepted in final form 19 September 2007
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ABSTRACT |
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13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption. intestinal folate absorption; proton-coupled folate transporters; heme carrier protein-1; hereditary folate malabsorption
Prior to the identification of HsPCFT, the reduced folate carrier (RFC), an anion exchanger, was often considered to represent the mechanism of intestinal folate absorption because it is highly expressed along the entire intestinal apical brush-border membrane and its expression is increased under conditions of folate deficiency (4, 15, 23). However, RFC-mediated transport has a pH optimum of 7.4 and a specificity profile (very low affinity for folic acid; influx Ki: 200 µM) that is very different from that observed for folate transport in the intestine and in cells and membrane vesicles of intestinal origin (low pH optimum, high affinity for folic acid; influx Km: 0.5 µM) (16, 30). Further evidence that the intestinal folate absorptive process is genetically distinct from RFC came from studies in which the low-pH folate transport activity was preserved in cell lines of intestinal and other tissue origins even when RFC was deleted from the genome or mutated with a loss of function (3, 29, 35, 36).
The present study addresses the functional properties and secondary structure of PCFT orthologs in the mouse [Mus musculus (Mm)PCFT] and rat [Rattus norvegicus (Rn)PCFT] along with the tissue expression pattern, specificity, and regulation in mice in response to dietary folate restriction. These experiments provide fundamental information necessary for the further characterization of the role of PCFT in mouse development and folate delivery to intestinal and other tissues and for the investigation of PCFT as a factor in the pathogenesis of cancer in mouse models.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Plasmid construction. Full-length MmPCFT and RnPCFT cDNA clones were obtained from Open Biosystems (Huntsville, AL). The open reading frames (ORFs) were PCR amplified with PfuUltra DNA polymerase (primers are shown in Table 1) and subsequently cloned into the BglII site of the pS64T vector for in vitro transcription of the capped sense MmPCFT and RnPCFT cRNA from a SP6 promoter using the mMESSAGE mMACHINE system (Ambion, Austin, TX) and into the BamHI site of pcDNA3.1(+) to generate pcDNA3.1(+)MmPCFT and pcDNA3.1(+)RnPCFT, respectively. MmPCFT and RnPCFT ORFs were tagged at either NH2- or COOH-termini with a hemaglutinin (HA) epitope by PCR (primers shown in Table 1) and cloned into pcDNA3.1(+) with the same strategy as above. cDNA inserts were verified by DNA sequencing at the Albert Einstein Cancer Center Genomics Shared Resource.
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Experiments in mice.
The impact of dietary folate restriction on PCFT mRNA levels was assessed in 3- to 4-mo-old male C57BL/6 specific pathogen-free mice. Animals were maintained on either folate-containing mouse chow (2 mg/kg folic acid) or a folic acid-free diet over 8 wk, after which they were killed in a CO2 chamber. The proximal small intestine was dissected and rinsed in ice-cold PBS (pH 7.4), and total RNA was isolated with TRIzol reagent (Invitrogen). Data on RFC and folate receptor (FR)-
levels in these animals have been previously reported (15).
For immunohistochemical experiments, C57BL/6 adult mice maintained on the usual mouse chow were killed, and segments of intestines were dissected, rinsed in ice-cold PBS (pH 7.4), and embedded in freezing embedding OCT medium (Andwin Scientific, Addison, IL) for cryosectioning at 5 µm thickness. Similar intestine segments were dissected for the isolation of total RNA.
Production of peptide antibody and immunohistochemistry. Antiserum specific for MmPCFT protein was custom made by Open Biosystems in rabbits using the peptide corresponding to amino acids 446–459 ([C]EKVNPHPEFQQFPQSP) of MmPCFT protein as the antigen. Antibodies specific for MmPCFT were affinity purified with the Sulfolink Trial Kit (Pierce). Rabbit anti-HA antibody was obtained from Sigma-Aldrich. Immunofluorescence was performed using either affinity-purified anti-MmPCFT or anti-HA antibodies and secondary swine anti-rabbit IgG antibody conjugated with FITC (DAKO, Carpinteria, CA). HeLa cells were stained with or without permeabilization in 0.2% Triton X-100 in PBS (pH 7.4) for 15 min, and fluorescence was visualized on an Olympus IX70 Inverted Epifluorescence Microscope (Center Valley, PA).
Preparation of cryosections of mouse tissues and immunochemistry with affinity-purified anti-MmPCFT antibody followed procedures previously described (31). The specificity of the anti-MmPCFT antibody was confirmed by blocking the immunohistochemical staining ofMmPCFT with the peptide antigen (200 µg/ml).
SDS-PAGE and Western blot analysis. HepG2 or HeLa cell membrane fractions were prepared using previously reported procedures (22). Protein samples (30 µg) were resolved by SDS-PAGE, and MmPCFT protein was detected by Western blot analysis using rabbit anti-MmPCFT peptide antibody solution (1:1,000 dilution) together with goat anti-rabbit horseradish peroxidase-conjugated IgG (1:5,000 dilution, Cell Signaling Technology, Danvers, MA).
Measurement of uptake of folates in HepG2 and HeLa cells.
Uptake of [3H]folic acid, [3H]MTX, or [3H](6S)5-methylTHF was assessed in HepG2 and HeLa cells grown in monolayer cultures at the bottom of liquid scintillation vials by modification of previously described procedures (22, 24). Initial uptake kinetics for tritiated folates were measured over a 2-min interval. This resulted in a small (30%) decrease in extracellular folate, due to cellular uptake, at the lowest concentration studied (0.1 µM). This decreased as the extracellular folate level increased and became negligible at concentrations >1.0 µM. This resulted in a small overestimation of Km; this would not be a factor in influx kinetic measurements obtained electrophysiologically.
To assess the effects of antiserum on folic acid uptake mediated by MmPCFT, stably transfected HepG2 cells were washed twice in HBS [20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM dextrose (pH 7.4)] and then incubated with 1 ml of HBS (pH 7.4) containing preimmune or anti-MmPCFT serum (1:50 dilution) at 37°C and pH 7.4 for 20 min. [3H]folic acid (0.5 µM) influx was then measured in MBS (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM dextrose) at pH 5.5 or 6.5 containing either preimmune or anti-MmPCFT serum (1:50 dilution) at 37°C for 2 min.
To examine the effects of sodium on the transport activities of MmPCFT or RnPCFT, [3H]folic acid (0.5 µM) uptake was assessed in MBS (pH5.5) over 2 min in which 140 mM NaCl was replaced by the same concentration of choline chloride. Uptake was normalized to protein content.
Electrophysiological analyses in Xenopus oocytes. Preparation and microinjection with 50 nl of water or PCFT cRNA (30–50 ng) into defolliculated Xenopus laevis oocytes followed previously described procedures; oocytes were kept at 17°C in horse serum medium [82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 2.3 mM CaCl2, 5 mM HEPES, and 5% horse serum (pH 7.5)], and electrophysiological recordings in a solution of 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Tris, and 5 mM MES were conducted 3–7 days after cRNA injection (10, 22). For the determination of Km values, currents were measured at different folate concentrations with the membrane potential clamped at –80 mV. Influx kinetic constants were obtained from the normalized currents from different experiments fitted to the following equation: I = (Imax x S)/(Km + S), where I is the current induced by a given substrate concentration (S) and Imax is the maximal current generated.
Northern blot analysis. A Northern blot containing polyA+ RNA (2 µg/lane) from 12 mouse tissues was obtained from Origene (Rockville, MD). [32P]dCTP-labeled cDNA probes were made from a MmPCFT cDNA segment (1094–1440 bp, GenBank Accession No. NM_026740) and hybridized to the membrane as previously described (22). After the membrane had been stripped, β-actin mRNA was probed as the loading control.
Total RNA (10 µg) isolated from different intestinal segments was resolved on a 1% denaturing agarose gel and transferred to a Nytran nylon membrane (Whatman, Florham Park, NJ). Northern blot hybridization was subsequently performed using the [32P]dCTP-labeled MmPCFT cDNA probe described above. 18S and 28S rRNAs were visualized after ethidium bromide staining as the loading control.
Quantitative RT-PCR. Total RNA isolated from mice fed folate-replete or folate-deficient diets, as previously reported (15) and described above, was reverse transcribed to cDNA with Superscript Reverse Transcriptase II (Invitrogen). Real-time PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and primers specific for MmPCFT (Table 1). GAPDH was simultaneously amplified with specific primers (Table 1) to normalize MmPCFT expression.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50 kDa (Fig. 1A). PCFT proteins are highly conserved in mammals. Based on sequence alignment analysis with ClustalW (13), the predicted MmPCFT and RnPCFT proteins share 95% amino acid identity to each other, 87% identity to HsPCFT, and >80% identity to their counterparts in other mammalian species, e.g., the dog (Canis familiaris; XP_548286
[GenBank]
), rhesus monkey (Macaca mulatta; XP_001106954), and domestic cow (Bos taurus; XM_588367) (Fig. 1, A and B). PCFT orthologs also appear to be present in nonmammalian vertebrates, e.g., the chicken [Gallus gallus (GgPCFT); XP415815], African clawed frog [X. laevis (XlPCFT); AAH77859
[GenBank]
], and zebrafish [Danio rerio (DrPCFT); AAH49421
[GenBank]
], all of which share >50% amino acid identity to mammalian PCFTs (Fig. 1B). No invertebrate homologs could be identified in the NCBI GenBank database with >30% amino acid identity to vertebrate PCFTs.
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Subcellular localization and topological properties of MmPCFT and RnPCFT proteins. Topology prediction programs [DAS, HMMTOP, PredictProtein, SOSUI, TMHMM, TMpred, TopPred, and Hydropathy Analysis (http://www.expasy.org/tools/#topology and http://www.tcdb.org/analyze.php)] were employed to characterize the PCFT secondary structure. The numbers of transmembrane domains (TMDs) varied with the different algorithms. Most of the programs predicted a polytopic integral membrane protein with 11 or 12 transmembrane segments, although TMpred predicted 10 transmembrane segments. A model with 12 TMDs, as predicted by DAS, HMMTOP, ProteinPrediction, and Hydropathy Analysis programs, is shown in Fig. 1A and indicates the high degree of homology within TMDs among human and rodent transporters.
An immunohistochemistry approach was used to assess the location of the NH2- and COOH-termini of rodent PCFT proteins. MmPCFT and RnPCFT were tagged with an HA epitope at either terminus, and the fusion proteins were subsequently immunodetected with an anti-HA antibody in HeLa cells transiently transfected with each construct with or without permeabilization before being immunostained. As shown in Fig. 2A, MmPCFT and RnPCFT HA fusion proteins could only be localized to the plasma membrane of permeabilized cells (top); these epitopes could not be stained in cells that were not permeabilized (bottom). Similar immunolocalization was also observed for wild-type MmPCFT expressed in HeLa cells (Fig. 2A, wild-type MmPCT), which could only be stained in permeabilized cells with an anti-MmPCFT peptide antibody that specifically recognizes an epitope in the COOH-terminus. These observations are consistent with an intracellular localization of the NH2- and COOH-termini of MmPCFT and RnPCFT and a predicted topology in which there are an even number of transmembrane segments, likely 12 transmembrane segments, as based on the topological analyses.
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Anti-MmPCFT serum, which specifically recognizes the COOH-terminus of the murine transporter, did not alter folic acid uptake in HepG2 cells stably transfected with MmPCFT, further supporting the intracellular localization of the COOH-terminus of MmPCFT protein (Fig. 2C).
Using anti-MmPCFT serum, MmPCFT protein was detected as broad bands in the Western blot analysis in both transiently transfected HeLa cells and stably transfected HepG2 cells with molecular weights higher than predicted (50 kDa) (Fig. 2D). This is consistent with the N-linked glycosylation site(s) in the extracellular loop between TMD 1 and TMD 2, which are highly conserved among rodent and human PCFTs (Fig. 1A) and have been confirmed in other studies by this laboratory (unpublished observations). In both HepG2 and HeLa transfectants, there was a band at 35 kDa detected in both mock- and MmPCFT-transfected HeLa and HepG2 cells that disappeared in the presence of blocking peptide. The origin of this band was not clear.
pH dependence of folate transport mediated by MmPCFT and RnPCFT.
The initial uptake of 0.5 µM [3H]folic acid (Fig. 3A), [3H]MTX (Fig. 3B), and [3H](6S)5-methylTHF (Fig. 3C) by MmPCFT and RnPCFT in stably transfected HepG2 cells was highly pH dependent, decreasing as pH was increased from pH 5.5 to 7.4. Whereas there was negligible folic acid and MTX transport at pH 7.0, there was residual activity for (6S)5-methylTHF at this pH. There was 50% of maximum activity for all these folate substrates at pH 6.0, the pH at the upper small intestinal absorptive surface (17). [3H]folic acid transport mediated by MmPCFT and RnPCFT in stably transfected HepG2 cells was not sodium dependent; influx of both species was the same in sodium-containing buffer and buffer in which sodium was replaced by choline (Fig. 3D).
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Folate influx kinetics. Initial rates of [3H]folic acid, [3H]MTX, and [3H](6S)5-methylTHF uptake in HepG2 cells stably transfected with cDNAs encoding MmPCFT and RnPCFT followed Michaelis-Menten kinetics (as illustrated for folic acid in Fig. 4A). Folic acid-induced currents in Xenopus oocytes injected with MmPCFT cRNA was saturable with increasing substrate concentrations at both pH 5.5 and 6.5 (Fig. 4B). Table 2 shows data comparing relative influx Km values for the various folates for mouse and rat PCFTs at pH 5.5 versus 6.5. Both MmPCFT and RnPCFT had comparable high affinities for folic acid at pH 5.5. When pH was increased from 5.5 to 6.5, the Km value for folic acid, (6S)5-methylTHF, and MTX was increased and Vmax was decreased. For instance, Km values for MTX increased from 1.0 ± 0.1 and 0.6 ± 0.1 µM to 4.6 ± 1.8 and 2.6 ± 0.8 µM for MmPCFT and RnPCFT, respectively, and Vmax decreased from 289.6 ± 48.8 and 281.3 ± 32.8 pmol·mg protein–1·2 min–1 to 137.9 ± 24.2 and 130.2 ± 16.1 pmol·mg protein–1·2 min–1, respectively. There was a lesser change in influx Km values for folic acid over this pH range, with no change for (6S)5-methylTHF as assessed by electrophysiological measurements in Xenopus oocytes (Table 2). Km values were somewhat overestimated using tritiated folates due to substrate depletion at low concentrations, which could account for differences in this parameter based on current measurements (see MATERIALS AND METHODS). In addition, these differences might be due to voltage-related changes in Km that occur during electrophysiological measurements.
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As shown in Fig. 4D, whereas uptake of 0.5 µM [3H]MTX at pH 5.5 mediated by MmPCFT and RnPCFT was nearly abolished by 50 µM nonlabeled MTX monoglutamate, the same concentration of MTX di-, tri-, tetra-, penta-, or hexaglutamate had no inhibitory effect at all, indicating that MmPCFT and RnPCFT are highly specific for monoglutamyl forms of folates.
Expression of PCFT mRNA and protein in mouse tissues.
MmPCFT mRNA, with a molecular size of 2.2 kb, was abundantly expressed in the small intestine, liver, and kidney and to a lesser extent in the brain, testis, skin, and stomach (Fig. 5A). There were low levels in the heart and lung, very low levels in the spleen and thymus, and no detectable MmPCFT mRNA in skeletal muscle. A shorter isoform of MmPCFT mRNA, with a molecular size of 1.3 kb, was also detected in the brain, kidney, liver, skin, small intestine, and stomach. Focusing on the intestinal tract, MmPCFT mRNA was highly expressed in the duodenum and proximal jejunum, with lower expression in the distal jejunum and even lower levels in the cecum and colon (Fig. 5B). There was just a trace of expression in the ileum and rectum.
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Alterations in MmPCFT mRNA levels in the proximal small intestine in response to dietary folate.
A previous study (15) demonstrated that folate deprivation in mice resulted in increased levels of both FR- and RFC mRNA. Using the RNA from these animals, we assessed the impact of folate deprivation on PCFT mRNA in the proximal small intestine in mice on a normal chow diet versus mice on a folate-deficient diet. As shown in Fig. 6, inset, MmPCFT mRNA levels were increased in each of 10 folate-deficient mice compared with folate-replete mice (n = 7) without an overlap in levels between the two groups. Overall, the MmPCFT mRNA level was increased by a factor of 13 in folate-deficient mice compared with folate-replete mice (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-glutamyl carboxypeptidase II to monoglutamate forms before transport into enterocytes can occur (7). Transport of folic acid, (6S)5-methylTHF, and MTX by mouse and rat PCFTs expressed in Xenopus oocytes was electrogenic, consistent with a net charge translocation during folate transport. The magnitude of the current was dependent on the extracellular folate concentration and on the extracellular pH. Alterations in protonation of the folate molecule do not occur over the pH range of 5.5 to 7.4 (20); however, at lower pHs, these measurements would be complicated by changes in substrate protonation, which, in turn, changes the concentration of the substrate recognized by the transporter. The change in current amplitude as a function of pH, under conditions in which changes in protonation of the folate molecule do not occur, suggest that pH changes not only alter the magnitude of the driving force for folate transport but may also titrate amino acids within PCFT that result in an alteration in the rate of transport. Studies are underway to identify the amino acids involved in these pH-dependent effects.
MmPCFT has been previously reported to be a heme carrier protein (HCP1) that transports heme with low affinity (125 µM) and independent of pH (14, 25). Within the context of that report, HCP1 was predicted to consist of nine transmembrane domains with the NH2- and COOH-termini located intracellularly and extracellularly, respectively. However, the present study, along with the previous report from this laboratory, indicates that transport mediated by this carrier is highly specific and pH dependent for folates, with an affinity at least two orders of magnitude higher than that reported for hemin. These data, together with topological prediction based on multiple hydropathy analyses, also support intracellular localization of both the NH2- and COOH-termini of MmPCFT and RnPCFT expressed in HeLa cells and an even number of transmembrane segments, most likely 12. However, in view of the limitations of the methodologies employed, confirmation of the localization of the NH2- and COOH-termini and other elements of this transporter will require further verification of the secondary structure.
The functional properties of MmPCFT and RnPCFT are consistent with the folate transport activities that have been reported for rat and mouse intestinal segments, brush-border membranes, and intestinal cell lines that have a low pH maximum (16, 29). Both transporters have a high level of folate transport activity at pH 6.0, the pH at the apical surface of the proximal small intestinal epithelium (17). MmPCFT mRNA and protein were highly expressed in the duodenum and upper jejunum with the protein exclusively localized to the brush-border membrane, consistent with its functional role as the rate-limiting first step in the absorption of dietary folates. The functional role of MmPCFT in intestinal folate absorption is further supported by the observation that MmPCFT mRNA levels in the murine proximal small intestine increased 13-fold in mice fed a folate-deficient diet. It is of interest that both RFC and FR- mRNA levels also increased in the same tissues from the same mice on a folate-deficient diet, indicating that expression of all these transporters is folate responsive (15). However, it is only PCFT that, in fact, mediates the translocation of folates at the acidic environment of the proximal small intestine at usual dietary folate levels. This conclusion is based on the observation that this gene is mutated in patients with hereditary folate malabsorption. Hence, this folate-responsive regulation of the PCFT gene is particularly important in folate deficiency states. It is possible, however, that some RFC-mediated transport can occur with the administration of pharmacological doses of folates, as in the treatment of hereditary folate malabsorption (6).
In addition to its high expression in the small intestine at the sites of folate absorption, PCFT is also highly expressed in other tissues. Very high expression of MmPCFT mRNA was observed in the liver and kidney, tissues that manifest a high level of folate transport activity at low pH (1, 9). The major fraction of folates in the glomerular filtrate, which is at pH 6.8 (11), are reabsorbed at the proximal tubules. At this pH, PCFT has substantial folate transport activity, suggesting that it may play a direct role in reabsorption of folates at this site. This is supported by the presence of substantial low pH folate transport activity in brush-border membrane vesicles from the rat kidney (1). The contribution of PCFT to folate transport in the liver, where it is also highly expressed, is not clear. Transport of folates into the liver from the arterial system occurs at neutral pH; however, the pH within the hepatic portal sinusoids, where folates are delivered via the portal system from the intestine to hepatocytes, is not known and may be acidic (9). Data for human and rodent PCFTs indicate that there is residual 5-methylTHF transport activity at pH 7.0 so that this carrier can operate, albeit less efficiently, at neutral pH.
Beyond transport at the level of the cell membrane, PCFT may have other functions. For instance, this carrier may be incorporated into membrane vesicles that accommodate folate receptors and transport folates by receptor-mediated endocytosis or transcytosis (33). FR- is highly expressed at the apical proximal renal tubule membrane and has been suggested to be a major route of folate reabsorption in the kidney (2). Folates bind to FRs that are anchored to the cell membrane via a glycosylphosphoinositol moiety. The resultant complex is internalized in a vesicle that traffics, intact, within the cytoplasm, where it is acidified, resulting in the release of the folate molecule from receptor (18, 19, 34). The mechanism by which folate is then exported from the vesicle has not been clarified. If PCFT is present in these endocytic vesicles, it could provide the route of folate transport into the cytoplasm using the outward transvesicular proton gradient. Such a mechanism has been proposed earlier, although the identity of a vesicular transporter that could function at low pH is not known (12, 21). This mechanism could also explain, in part, why patients with hereditary folate malabsorption have a defect in folate transport into the central nervous system where the pH is neutral at the transport site but FR- is highly expressed at the choroid plexus (32). The detection of substantial levels of PCFT mRNA in the mouse brain is consistent with this hypothesis.
Analysis of the NCBI GenBank genome database shows that PCFT is highly conserved in other mammalian species at the level of both the gene and primary protein structures, e.g., in the dog, rhesus monkey, and domestic cow and, to a lesser extent, in nonmammalian vertebrates such as birds, amphibians, and fish. Together with its functional conservation in humans, mice, and rats, this suggests that PCFT is evolutionarily conserved for intestinal folate absorption in mammalian species and likely plays this role in nonmammalian vertebrates as well. PCFT does not appear to be conserved in invertebrates according to a protein-protein BLAST search of the NCBI GenBank database using vertebrate PCFTs as queries, suggesting that this gene might have originated and evolved in vertebrates.
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FOOTNOTES |
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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.
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REFERENCES |
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2. Birn H, Spiegelstein O, Christensen EI, Finnell RH. Renal tubular reabsorption of folate mediated by folate binding protein 1. J Am Soc Nephrol 16: 608–615, 2005.
3. Chattopadhyay S, Zhao R, Krupenko SA, Krupenko N, Goldman ID. The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line. Mol Cancer Ther 5: 438–449, 2006.
4. Chiao JH, Roy K, Tolner B, Yang CH, Sirotnak FM. RFC-1 gene expression regulates folate absorption in mouse small intestine. J Biol Chem 272: 11165–11170, 1997.
5. Fry DW, Yalowich JC, Goldman ID. Rapid formation of poly-gamma-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascited tumor cell in vitro. J Biol Chem 257: 1890–1896, 1982.
6. Geller J, Kronn D, Jayabose S, Sandoval C. Hereditary folate malabsorption: family report and review of the literature. Medicine (Baltimore) 81: 51–68, 2002.[CrossRef][Medline]
7. Halsted CH, Ling EH, Luthi-Carter R, Villanueva JA, Gardner JM, Coyle JT. Folylpoly-gamma-glutamate carboxypeptidase from pig jejunum. Molecular characterization and relation to glutamate carboxypeptidase II. J Biol Chem 273: 20417–20424, 1998.
8. Henderson GB, Strauss BP. Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells. Cancer Res 50: 1709–1714, 1990.
9. Horne DW. Transport of folates and antifolates in liver. Proc Soc Exp Biol Med 202: 385–391, 1993.[Medline]
10. Jansen M, Akabas MH. State-dependent cross-linking of the M2 and M3 segments: functional basis for the alignment of GABAA and acetylcholine receptor M3 segments. J Neurosci 26: 4492–4499, 2006.
11. Jaramillo-Juarez F, Aires MM, Malnic G. Urinary and proximal tubule acidification during reduction of renal blood flow in the rat. J Physiol 421: 475–483, 1990.
12. Kamen BA, Smith AK, Anderson RGW. The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro. J Clin Invest 87: 1442–1449, 1991.[Web of Science][Medline]
13. Lassmann T, Sonnhammer EL. Kalign, Kalignvu and Mumsa: web servers for multiple sequence alignment. Nucleic Acids Res 34: W596–W599, 2006.
14. Latunde-Dada GO, Takeuchi K, Simpson RJ, McKie AT. Haem carrier protein 1 (HCP1): expression and functional studies in cultured cells. FEBS Lett 580: 6865–6870, 2006.[CrossRef][Web of Science][Medline]
15. Liu M, Ge Y, Cabelof DC, Aboukameel A, Heydari AR, Mohammad R, Matherly LH. Structure and regulation of the murine reduced folate carrier gene: identification of four noncoding exons and promoters and regulation by dietary folates. J Biol Chem 280: 5588–5597, 2005.
16. Mason JB, Rosenberg IH. Intestinal absorption of folate. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1994, p. 1979–1995.
17. McEwan GT, Daniel H, Fett C, Burgess MN, Lucas ML. The effect of Escherichia coli STa enterotoxin and other secretagogues on mucosal surface pH of rat small intestine in vivo. Proc R Soc Lond B Biol Sci 234: 219–237, 1988.[Medline]
18. Murphy RF, Powers S, Cantor CR. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol 98: 1757–1762, 1984.
19. Paulos CM, Reddy JA, Leamon CP, Turk MJ, Low PS. Ligand binding and kinetics of folate receptor recycling in vivo: impact on receptor-mediated drug delivery. Mol Pharmacol 66: 1406–1414, 2004.
20. Poe M. Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate. J Biol Chem 252: 3724–3728, 1977.
21. Prasad PD, Mahesh VB, Leibach FH, Ganapathy V. Functional coupling between a bafilomycin A1-sensitive proton pump and a probenecid-sensitive folate transporter in human placental choriocarcinoma cells. Biochim Biophys Acta Mol Cell Res 1222: 309–314, 1994.[Medline]
22. Qiu A, Jansen M, Sakaris A, Min SH, Chattopadhyay S, Tsai E, Sandoval C, Zhao R, Akabas MH, Goldman ID. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 127: 917–928, 2006.[CrossRef][Web of Science][Medline]
23. Said HM. Recent advances in carrier-mediated intestinal absorption of water-soluble vitamins. Annu Rev Physiol 66: 419–446, 2004.[CrossRef][Web of Science][Medline]
24. Sharif KA, Goldman ID. Rapid determination of membrane transport parameters in adherent cells. Biotechniques 28: 926–928, 930, and 932, 2000.[Web of Science][Medline]
25. Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, Khan Y, Warley A, McCann FE, Hider RC, Frazer DM, Anderson GJ, Vulpe CD, Simpson RJ, McKie AT. Identification of an intestinal heme transporter. Cell 122: 789–801, 2005.[CrossRef][Web of Science][Medline]
26. Sierra EE, Goldman ID. Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function. Biochem Pharmacol 55: 1505–1512, 1998.[CrossRef][Web of Science][Medline]
27. Smith SB, Kekuda R, Gu X, Chancy C, Conway SJ, Ganapathy V. Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina. Invest Ophthalmol Vis Sci 40: 840–848, 1999.
28. Stover PJ. Physiology of folate and vitamin B12 in health and disease. Nutr Rev 62: S3–S12, 2004.[CrossRef][Web of Science][Medline]
29. Wang Y, Rajgopal A, Goldman ID, Zhao R. Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function. Am J Physiol Cell Physiol 288: C65–C71, 2005.
30. Wang Y, Zhao R, Goldman ID. Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier. Clin Cancer Res 10: 6256–6264, 2004.
31. Wang Y, Zhao R, Russell RG, Goldman ID. Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis. Biochim Biophys Acta 1513: 49–54, 2001.[Medline]
32. Weitman SD, Weinberg AG, Coney LR, Zurawski VR Jr, Jennings DS, Kamen BA. Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis. Cancer Res 52: 6708–6711, 1992.
33. Williams WM, Huang KC. Renal tubular transport of folic acid and methotrexate in the monkey. Am J Physiol Renal Fluid Electrolyte Physiol 242: F484–F490, 1982.
34. Yang J, Chen H, Vlahov IR, Cheng JX, Low PS. Characterization of the pH of folate receptor-containing endosomes and the rate of hydrolysis of internalized acid-labile folate-drug conjugates. J Pharmacol Exp Ther 321: 462–468, 2007.
35. Zhao R, Gao F, Hanscom M, Goldman ID. A prominent low-pH methotrexate transport activity in human solid tumor cells: Contribution to the preservation of methotrexate pharmacological activity in HeLa cells lacking the reduced folate carrier. Clin Cancer Res 10: 718–727, 2004.
36. Zhao R, Hanscom M, Goldman ID. The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells. Biochim Biophys Acta 1715: 57–64, 2005.[Medline]
37. Zhao R, Min SH, Qiu A, Sakaris A, Goldberg GL, Sandoval C, Malatack JJ, Rosenblatt DS, Goldman ID. The spectrum of mutations in the PCFT gene, coding for an intestinal folate transporter, that are the basis for hereditary folate malabsorption. Blood 110: 1147–1152, 2007.
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