A novel deletion mutation in the proton-coupled folate transporter (PCFT; SLC46A1) in a Nicaraguan child with hereditary folate malabsorption

Hereditary folate malabsorption (OMIM 229050) is a rare autosomal recessive disorder caused by loss-of-function mutations in the proton-coupled folate transporter gene (pcft/SLC46A1) resulting in impaired folate transport across the intestine and into the central nervous system. We report a novel, homozygous, deletion mutation in a child of Nicaraguan descent in exon 2 (c.558–588 del, ss778190447) at amino acid position I188 resulting in a frameshift with a premature stop.     

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high K_m) for the substrate and by decreasing the number of transporter molecules (low V_max) on the colon luminal surface. The decreased transport activity at the CAM was associated with down‐regulation of the proton‐coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. J. Cell. Physiol. 226: 579–587, 2011. © 2010 Wiley‐Liss, Inc.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 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.     

Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.     

Identification and functional impact of homo-oligomers of the human proton-coupled folate transporter

The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His(10) epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a "dominant-positive" phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.     

Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant (K(m)) for the former (2.4 microM) than for the latter (5.7 microM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the K(m) values were 1.2 and 5.8 microM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.     

Decreased placental folate transporter expression and activity in first and second trimester in obese mothers

Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8–22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (−19%) and FR-α (−17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by−52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

Biochemical and molecular mechanisms of folate transport in rat pancreas; interference with ethanol ingestion

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency which is due, in part to folate malabsorption. The present study deals with the mechanistic insights of reduced folate absorption in pancreas during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and the mechanisms of alcohol associated reduced folate uptake was studied in pancreas. The folate transport system in the pancreatic plasma membrane (PPM) was found to be acidic pH dependent one. The transporters proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are involved in folate uptake across PPM. The folate transporters were found to be associated with lipid raft microdomain of the PPM. Ethanol ingestion decreased the folate transport by reducing the levels of folate transporter molecules in lipid rafts at the PPM. The decreased transport efficiency of the PPM was reflected as reduced folate levels in pancreas. The chronic ethanol ingestion led to decreased pancreatic folate uptake. The decreased levels of PCFT and RFC expression in rat PPM were due to decreased association of these proteins with lipid rafts (LR) at the PPM.     

A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding

Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx K(t) was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx K(t) mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a K(i) for WT-PCFT comparable to its influx K(t). However, the methotrexate influx K(i) for P425R-PCFT was ～15-fold higher than the WT-PCFT influx K(t) and threefold higher than the methotrexate influx K(t) for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.     

Substituted Cysteine Accessibility Reveals a Novel Transmembrane 2–3 Reentrant Loop and Functional Role for Transmembrane Domain 2 in the Human Proton-coupled Folate Transporter

The proton-coupled folate transporter (PCFT) is a folate-proton symporter highly expressed in solid tumors that can selectively target cytotoxic antifolates to tumors under acidic microenvironment conditions. Predicted topology models for PCFT suggest that the loop domain between transmembrane domains (TMDs) 2 and 3 resides in the cytosol. Mutations involving Asp-109 or Arg-113 in the TMD2-3 loop result in loss of activity. By structural homology to other solute carriers, TMD2 may form part of the PCFT substrate binding domain. In this study we mutated the seven cysteine (Cys) residues of human PCFT to serine, creating Cys-less PCFT. Thirty-three single-Cys mutants spanning TMD2 and the TMD2-3 loop in a Cys-less PCFT background were transfected into PCFT-null HeLa cells. All 33 mutants were detected by Western blotting, and 28 were active for [³H]methotrexate uptake at pH 5.5. For the active residues, we performed pulldown assays with membrane-impermeable 2-aminoethyl methanethiosulfonate-biotin and streptavidin beads to determine their aqueous-accessibilities. Multiple residues in TMD2 and the TMD2-3 loop domain reacted with 2-aminoethyl methanethiosulfonate-biotin, establishing aqueous accessibilities. Pemetrexed pretreatment inhibited biotinylation of TMD2 mutants G93C and F94C, and biotinylation of these residues inhibited methotrexate transport activity. Our results suggest that the TMD 2–3 loop domain is aqueous-accessible and forms a novel reentrant loop structure. Residues in TMD2 form an aqueous transmembrane pathway for folate substrates, and Gly-93 and Phe-94 may contribute to a substrate binding domain. Characterization of PCFT structure is essential to understanding the transport mechanism including the critical determinants of substrate binding.     

Random mutagenesis of the proton-coupled folate transporter (SLC46A1), clustering of mutations, and the bases for associated losses of function

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5'-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.     

Structural basis for recognition and transport of folic acid in mammalian cells

Structural studies on mammalian vitamin transport lag behind other metabolites. Folates, also known as B9 vitamins, are essential cofactors in one-carbon transfer reactions in biology. Three different systems control folate uptake in the human body; folate receptors function to capture and internalise extracellular folates via endocytosis, whereas two major facilitator superfamily transporters, the reduced folate carrier (RFC; SLC19A1) and proton-coupled folate transporter (PCFT; SLC46A1) control the transport of folates across cellular membranes. Targeting specific folate transporters is being pursued as a route to developing new antifolates with improved pharmacology. Recent structures of the proton-coupled folate transporter, PCFT, revealed key insights into antifolate recognition and the mechanism of proton-coupled transport. Combined with previously determined structures of folate receptors and new predictions for the structure of the RFC, we are now able to develop a structure-based understanding of folate and antifolate recognition to accelerate efforts in antifolate drug development.     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.