From the Departments of Medicine and Molecular Pharmacology, and the Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in MTXrA-E45K cells was characterized by a 12- and 3.6-fold decrease in influx Vmax and Kt respectively, relative to L1210 cells. The folic acid influx Ki in L1210 cells was more than 50-fold greater than that of MTX based upon inhibition of 5-CHO-THF influx. In comparison, the mutated carrier had comparable affinities for folic acid and MTX in MTXrA-E45K cells due to a 7-fold decrease in the folic acid influx Ki and 7-fold increase in the MTX influx Ki. Transport via native RFC1 is inhibited by a variety of anions in L1210 cells associated with an increase in influx Kt. However, influx of 5-CHO-THF in MTXrA-E45K cells in a HEPES buffer (9 mM chloride) was decreased by 70% due to a 3-fold fall in the Vmax. In the complete absence of chloride (K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10% that in HBS buffer. 5-CHO-THF influx was restored by addition of chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP which were all inhibitory over a broad range of concentrations.
The data suggest that substitution of a positive for a negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when cells are placed under selective pressure with antifolates that utilize RFC1 as the major route of entry into mammalian cells.
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INTRODUCTION |
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Folates are key elements in mammalian biosynthetic processes, and even subtle folate deficiency has been implicated in developmental defects (1), pre-neoplastic or neoplastic lesions (2), and disorders of homocysteine metabolism associated with increased risk for cardiovascular diseases (3, 4). Since diffusion of folates into cells is a slow process, particularly at their low levels present in blood under physiological conditions, cells must meet their folate requirement through facilitated or energy-requiring transport processes (5-7).
The major route of folate delivery into cells is the reduced folate carrier (RFC1),1 a typical facilitative transmembrane protein with 12 predicted transmembrane domains. Uptake of MTX is also mediated by RFC1 and impaired MTX transport is a common mechanism of resistance in clinical regimens (8, 9). Transport-related drug resistance has been associated with decreased expression of RFC1 or/and loss of function of the carrier (9). The recent cloning of this anion-exchanging transporter (10-15) now allows the development of an understanding of its structural-functional relationships, in particular the regions that are involved in folate binding and translocation. Of particular value in deciphering these properties are the functional changes that occur when the carrier is mutated under antifolate selective pressure in the presence of a variety of different folate substrates.
This laboratory has initiated studies using chemical mutagenesis to define the structural requirements for the transport of folate compounds in murine leukemia cells. In the first report on this work a substitution of asparagine for serine at amino acid 46 in the first predicted transmembrane domain was described in cells that were selected in the presence of MTX with 5-CHO-THF as the sole folate source (16). This mutation resulted in markedly impaired MTX transport, whereas sufficient transport capacity for 5-CHO-THF was retained to minimally affect the growth requirement for this essential substrate.
In the present report a second mutation is characterized in an adjacent amino acid, 45, that occurred in cells subjected to MTX selective pressure with folic acid as the sole folate source. This substitution of a lysine for glutamate resulted in a marked loss of antifolate and reduced folate transport but enhanced carrier-mediated transport of folic acid. This mutation also resulted in a qualitative change in the effects of anions on the carrier-mediated transport of folates, producing an altered carrier that absolutely requires, rather than is only inhibited by, the presence of extracellular inorganic anions for folate entry into cells. These observations, along with additional work from this (16) and other laboratories (17), indicate that the first putative transmembrane domain is an important site of interactions between RFC1 and folates that affects substrate binding and translocation and is a critical region for the development of mutations, in the presence of antifolate selective pressure, that produce highly diverse functional consequences.
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MATERIALS AND METHODS |
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Chemicals-- [3',5',7-3H](6S)-5-CHO-THF and [3',5',7-3H](6S)-5-CH3THF were obtained from Moravek Biochemicals (Brea, CA), and [3',5',7-3H]MTX was obtained from Amersham Pharmacia Biotech. Folates were purified by high performance liquid chromatography prior to use (18). Trimetrexate (TMQ) was a gift from Warner-Lambert and Parke-Davis (Ann Arbor, MI). All other reagents were of the highest purity available from various commercial sources.
Cell Culture Conditions and Growth Studies-- L1210 murine leukemia cells were grown in RPMI 1640 medium containing 2.3 µM folic acid, supplemented with 5% bovine calf serum (HyClone), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a humidified atmosphere of 5% CO2. Prior to assessment of folate growth requirement, cells were grown for 1-2 weeks in folate-free RPMI 1640 growth medium supplemented with 5% dialyzed bovine calf serum (HyClone), 200 µM glycine, 100 µM adenosine, and 10 µM thymidine (GAT) to deplete the endogenous folates. For analyses of growth requirement and inhibition, cells were grown in 96-well plates (1 × 105 cells/ml), exposed continuously to the appropriate concentrations of MTX, TMQ, folic acid, or 5-CHO-THF for 72 h following which cell numbers were determined by hemocytometer count, and viability assessed by trypan blue exclusion. G418 was omitted from the medium in growth studies with transfectants (see below).
Northern Analyses--
Total RNA was isolated using the TRIzol
reagent (Life Technologies, Inc.). RNA (20 µg) was resolved by
electrophoresis on 1% agarose gels containing formaldehyde. Transfer
and hybridization were performed as described previously (19).
Transcripts were quantitated by PhosphorImager analysis of the
hybridization signals and normalized to -actin.
Transport Buffers-- The following buffers were employed to determine folate transport: HEPES-buffered saline (HBS), 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM glucose, pH 7.4; HEPES buffer, 190 mM HEPES, 5 mM KCl, 2 mM MgCl2, 5 mM glucose, pH 7.4; Mg2+-free HBS, 20 mM HEPES, 140 mM NaCl, 5 mM glucose, pH 7.4; and K+-HEPES-sucrose buffer, 20 mM HEPES, 235 mM sucrose, pH 7.4 (adjusted with KOH). All buffers were adjusted to an osmolality of 290 ± 5 mmol/kg.
Transport Studies-- Influx measurements were performed by methods described previously (19) with minor modifications. Briefly, exponentially growing cells were harvested, washed twice, and resuspended in the appropriate buffer to a density of 1.5 × 107 cells/ml. Cell suspensions were incubated at 37 °C for 25 min following which uptake was initiated by the addition of radiolabeled folate, and samples were removed at indicated times. Uptake was terminated by injection of 1 ml of the cell suspension into 10 ml of ice-cold HBS. Cells were collected by centrifugation, washed twice with ice-cold HBS, and processed for measurement of intracellular radioactivity (19). Initial rates were established over an interval in which radiotracer folate uptake was linear as a function of time with an extrapolated ordinate intercept at time 0 near the point of origin. Prior to transport studies with transfectants (see below), cells were expanded for only 3-7 doublings without further addition of G418 in order to ensure that expression of the mutated RFC1 was maintained.
Isolation of the MTX Transport-deficient Clonal L1210-C8a Cell Line-- L1210 cells grown in complete RPMI medium were treated with 0.4 mM N-methyl-N-nitrosourea for 12 h to achieve about 10% cell survival (20). After cells were washed to remove the mutagen, they were placed in 24-well plates at a density of 2 × 105cells/ml and allowed to grow for 3 days. The cells (0.5 ml) from each well were then seeded in fresh complete RPMI medium containing 200 nM MTX and grown for 2 additional weeks. The surviving cells were maintained in this selection medium for another 3 weeks, after which they were plated in complete RPMI 1640 containing 0.5% soft agar. After an additional 2 weeks, individual clones were picked up and expanded, and MTX influx and growth inhibition as well as 5-CHO-THF and folic acid growth requirements were determined. One clone, L1210-C8a, with markedly impaired MTX transport and a ~2-fold decrease in the folic acid growth requirement, was selected for further study. The L1210-C8a cell line has been maintained in drug-free complete RPMI 1640 medium for 6 months and has maintained stable phenotypes for MTX transport and growth inhibition as well as growth requirements for folic acid and 5-CHO-THF.
Cloning of the Mutated Reduced Folate
Carrier--
Poly(A)+ mRNA was purified using
Dynabeads mRNA DIRECT kit (Dynal). The first DNA strand synthesis
was carried out with a Superscript Reverse Transcriptase according to
the manufacturer's protocol (Life Technologies, Inc.). The RFC1
protein coding sequence was amplified with Pfu polymerase
(Stratagene) by utilizing oligonucleotide primers that flank the coding
region of RFC1 (upstream primer, at nucleotide 46 from the
translation start codon 5'-GCGGATCCTGGAGTGTCATCTTGG-3' and downstream
primer at nucleotide +82 from translation stop codon
5'-GCCTCGAGCTGGTTCAGGTGGAGT-3'). Two 8-base pair linkers were
introduced into the primers so that both BamHI and
XhoI restriction sites were created in the PCR products to
facilitate directional cloning. The PCR amplifications were performed
for 35 cycles of 45 s at 95 °C, 45 s at 65 °C, and 3 min at 72 °C. The 1682-base pair-long predicted PCR product was
purified on an agarose gel (Qiagen), cloned into a pCR-Blunt vector
(Invitrogen), and sequenced on automated sequencer models ABI 373A and
ABI 373 from Perkin-Elmer in the sequencing facility of the Albert
Einstein College of Medicine Comprehensive Cancer Center.
Transfections-- RFC1-E45K cDNA was excised from the pCR-blunt vector (see above) by a double digestion with BamHI and XhoI and recloned into an expression vector pCDNA3.1(+) (Invitrogen) with the same restriction sites. MTXrA (1 × 107 cells), in which the endogenous RFC1 is not functional (21, 22), was electroporated (250 V, 200 microfarads) with 50 µg of nonlinearized pCDNA3.1(+) harboring the mutated RFC1-E45K cDNA in a final volume of 800 µl of serum-free RPMI 1640 medium. Cells were then diluted in 20 ml of complete RPMI 1640 medium, allowed to recover for 48 h, adjusted to 2 × 105 cells/ml in medium containing G418 (750 µg/ml of active drug) and then distributed into 96-well plates at approximately 4 × 104 cells/well. G418-resistant clones were isolated by limiting dilution and then expanded for further studies. One of the clones, MTXrA-E45K, which exhibited the highest level of RFC1 mRNA was selected for further studies.
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RESULTS |
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Characterization of MTX Transport-defective L1210-C8a Cells-- A number of MTX transport-defective L1210 variants were isolated with folic acid as the sole folate source after mutagenesis with N-methyl-N-nitrosourea and selection in the presence of MTX. This report focuses on one of the clonal mutant lines, L1210-C8a. The mRNA level of RFC1 in L1210-C8a was indistinguishable from that of L1210 cells (Fig. 1, 1st two lanes; the last two lanes will be discussed below). However, initial rates of uptake of MTX and 5-CHO-THF at a concentration of 1 µM in this cell line was only 8 and 15%, respectively, that of parental L1210 cells (Table I). The MTX growth inhibition IC50 and 5-CHO-THF growth requirement EC50 values concomitantly increased by factors of 19 and 10 in L1210-C8a cells, respectively, as compared with L1210 cells, consistent with their impaired delivery into cells. In contrast, influx of folic acid was unchanged or slightly increased, and the folic acid growth requirement was decreased as reflected in the reduced EC50 value in L1210-C8a cells.
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Characterization of the RFC1 Mutation in L1210-C8a Cells-- Studies were undertaken to characterize the genetic changes in RFC1 responsible for the altered transport phenotype in L1210-C8a cells. cDNAs representing the entire coding region of RFC1 were isolated from these cells by reverse transcriptase-PCR, cloned, and sequenced. The coding sequence was identical to that reported for murine RFC1 (GenBankTM accession number U32469) except for a G to A substitution at nucleotide position 133 when counting from the translation initiation codon. This single purine nucleotide change resulted in a substitution of lysine for glutamate at amino acid residue 45. The mutation was verified by sequencing another two randomly picked RFC1 cDNA clones.
Characterization of the Functional Properties of the Mutated Carrier-- To study the functional properties of the glutamate to lysine mutation, uncomplicated by other possible changes in L1210-C8a cells, the cDNA of the mutated carrier, designated RFC1-E45K, was transfected into MTXrA cells in which the endogenous carrier is not functional (21, 22), and a clonal derivative, MTXrA-E45K, was selected for further studies. The level of 1.9-kb RFC1 mRNA derived from the expression vector in the transfected cell line was 9 times greater than that of the endogenous 2.3-kb RFC1 transcript in MTXrA cells, from which it was derived, and 4.5 times greater than that from L1210 cells based on three separate Northern analyses (Fig. 1, the last 3 lanes). As depicted in Fig. 2, influx of folic acid (upper panel) was increased by a factor of 2, whereas influx of MTX (middle panel) and 5-CHO-THF (lower panel) were decreased by factors of 7 and 4, respectively, in MTXrA-E45K cells as compared with L1210 cells. The MTX and 5-CHO-THF transport activity measured in the transfectant could be attributed almost entirely to the RFC1-E45K since influx of MTX and 5-CHO-THF in MTXrA cells was less than 2% that of L1210 cells (not shown). Likewise the increase in folic acid influx in MTXrA-E45K cells was attributed entirely to the mutated carrier, since influx of folic acid in L1210 and MTXrA cells was comparable (data not shown).
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Influx Kinetics in MTXrA-E45K Cells-- To explore the basis for the alterations in transport mediated by the mutated RFC1, influx kinetics for 5-CHO-THF were determined in MTXrA-E45K and L1210 cells. Influx of 5-CHO-THF in both cell lines followed Michaelis-Menten kinetics, and the Kt and Vmax values were decreased in MTXrA-E45K cells by factors of 3.6 and 12, respectively, as compared with L1210 cells (Table II). Initial evaluation of MTX-influx kinetics in MTXrA-E45K cells suggested that the Kt was markedly increased compared with L1210 cells, and accurate measurements of kinetic parameters could not be obtained. Determination of folic acid influx via the carrier was also complicated by this consideration along with a very large component mediated by other route(s) (23). Indeed, influx of folic acid in L1210 and MTXrA cells was identical, and MTX had little or no inhibitory effect on folic acid influx in either cell lines. Hence, accurate direct measurement of influx kinetics for folic acid mediated by the mutated RFC1 was not possible. To circumvent these obstacles, Ki values for MTX and folic acid influx were obtained based upon inhibition of 5-CHO-THF influx. Dixon plot analyses for folic acid and MTX inhibition are illustrated in Fig. 3. As expected, inhibition of 5-CHO-THF influx by MTX and folic acid was competitive. The Ki for MTX was increased by a factor of 7, whereas the Ki for folic acid influx was decreased by a factor of 7 in the MTXrA-E45K transfectant as compared with L1210 cells (Table II). Interestingly, the inhibitor constants for MTX and folic acid influx in MTXrA-E45K are similar, whereas there is a more than 50-fold difference in L1210 cells favoring MTX.
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Growth Inhibition by MTX and Folate Growth Requirement in MTXrA-E45K Cells-- Consistent with low MTX influx in MTXrA-E45K cells, the IC50 for MTX was still ~35-fold greater than that in L1210 cells and was decreased by a factor of only 3 as compared with MTXrA (Fig. 4, upper panel). Expression of the mutated carrier in MTXrA cells decreased the 5-CHO-THF growth dependence by a factor of 25, approaching the EC50 of L1210 cells (middle panel). The decrease in the folic acid influx Ki along with the overexpression of the carrier in MTXrA-E45K cells translated into a 20-fold decrease in folic acid growth requirement relative to L1210 cells (lower panel).
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Anion Dependence of Folate Influx in MTXrA-E45K Cells-- Influx of folates via RFC1 in L1210 cells is influenced by the anionic composition of the extracellular milieu. Inorganic and organic anions are inhibitory to this process, and the transmembrane organic anion gradient has been implicated as a source of energy that sustains uphill folate transport into cells (24-26). Since a negatively charged glutamate residue was replaced with a positively charged lysine in the mutated carrier RFC1-E45K, studies were undertaken to assess the consequences of this structural alteration on the effect of anions on folate transport. In contrast to the near 2-fold stimulation of influx seen in L1210 cells when buffer NaCl was replaced iso-osmotically with HEPES (HEPES buffer), 5-CHO-THF influx was decreased by 70% in MTXrA-E45K cells under these conditions (Fig. 5). These changes were not related to alterations in extracellular Na+ composition since influx in both cell lines was unchanged by substitution of Na+ with Li+ (not shown). Therefore either chloride stimulated or HEPES inhibited the activity of RFC1-E45K.
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DISCUSSION |
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This laboratory has initiated studies that characterize the relationship between structure and function of the murine RFC1 using chemical mutagens to generate a broad spectrum of mutations within the carrier (16). A variety of selective pressures has been applied to simulate specific conditions that occur in vivo and in vitro in order to generate a diversity of functional changes that optimize identification of the regions of RFC1 that are key determinants of carrier mobility and substrate binding.
In an initial report, cells were selected in the presence of MTX with 5-CHO-THF as the folate substrate to mimic conditions in which tumor cells develop resistance to antifolates in vivo. A drug-resistant line was identified with a serine to asparagine substitution at amino acid 46 (illustrated in Fig. 7) that resulted in a marked reduction in the transport of MTX, but with a much lesser decrease in 5-CHO-THF and 5-CH3-THF influx (16). The relative conservation of tetrahydrofolate cofactor transport explained, in part, how tumors that develop resistance to MTX in vivo can meet their requirement for folates when the major circulating folate in the blood of man and rodents is 5-CH3-THF.
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This report focuses on the development of L1210 leukemia cell lines with acquired resistance to MTX under conditions in which folic acid was the sole folate source. In L1210 cells, as well as other mammalian cells, the affinity of RFC1 for folic acid is very low (5, 24, 29, 30) and the majority of influx is mediated by different route(s) (23, 31). Hence the loss of RFC1 activity minimally affects the viability of cells in a growth medium supplemented with 2.3 µM folic acid (32-34), and all mutants with impaired MTX transport can be cloned and the properties of the carrier characterized. A number of MTX transport-defective L1210 clones were isolated under these conditions. This report describes a mutation isolated from one of these clonal lines, L1210-C8a, that resulted in a glutamate to lysine substitution at amino acid 45, one amino acid away from the mutation described in our recent report (16).
Alterations in transport activity mediated by RFC1 can be associated with changes in the level of carrier expression or mutations in the carrier that result in alterations in the influx Kt and/or Vmax. In the previous report, alterations in the transport of MTX and reduced folates produced by the substitution of asparagine for serine at amino acid 46 were consistent with markedly different substrate-dependent changes in the mobility of the loaded carrier with minimal or no changes in the substrate binding affinities (16). In the present study the glutamate to lysine substitution at amino acid 45 produced a carrier species with a marked reduction in the influx Vmax for 5-CHO-THF. Indeed, because of the apparent 4.5-fold higher level of carrier expression in the transfectant (assuming comparable levels of translation), the mobility of the loaded mutated carrier is likely to be considerably lower than the 12-fold measured decrease in the Vmax in the transfectant relative to the wild type cells. This was, however, associated with a 3-fold decrease in the influx Kt; hence there was enhancement of binding, but to a much smaller extent than the decline in Vmax.
The Vmax for MTX and folic acid influx mediated by the mutated carrier could not be measured directly, but inhibition of 5-CHO-THF influx by these folates could be quantitated. By this approach the influx Ki for folic acid decreased by a factor of 7, and the Ki for MTX increased by a comparable extent in the transfectant relative to L1210 cells. This suggests that the Kt of the mutated carrier for both these substrates is essentially the same unlike parental L1210 cells in which the Kt values differ by a factor of at least 50. An increase in folic acid influx in the MTXrA-E45K cells is attributed to both the increased affinity of the mutated carrier for folic acid and the 4.5-fold higher level of carrier expression in the transfectant relative to L1210 cells. That the measured folic acid influx in MTXrA-E45K cells was much smaller than expected is attributed to the fact that only a small component of the influx in L1210 cells is mediated by the carrier (23). Hence, what might be expected to be a nearly 30-fold increase in influx via RFC1 is translated into only a 2-fold change in the total influx.
Phenyalanine substitution for isoleucine at amino acid 48 resulted in an even larger decrease in the folic acid influx Kt, although in this case MTX influx was largely preserved (17).2 Glutamate 45 and isoleucine 48 are separated by two residues but are likely to be in proximity in an aqueous pocket in the cell membrane since 3.6 amino acids are required to create a full turn within the helix. Hence, both sites appear to play a key role in folic acid binding. A tryptophan to glycine substitution in the predicted third transmembrane domain also markedly increased carrier affinity for folic acid (17)2 raising the possibility that there may be interactions between these regions in the folded carrier protein. In any event, these observations suggest that the first transmembrane domain plays a key role in determining the functional properties of RFC1 and is likely to be a mutation cluster region when cells are placed under antifolate selective pressure. These mutations are illustrated in Fig. 7, the predicted secondary structure of RFC1.
In a recent report, a single amino acid change in the predicted external loop between the seventh and eighth transmembrane domains of RFC1 (also indicated in Fig. 7) from two different murine tumor cell lines was associated with a 3-fold difference in the influx Km without a change in Vmax (35). The present study and another report (17) indicate that the binding of folates to RFC1 is influenced not only by amino acid residues in external loops but also by interactions with residues well within the transmembrane domain. This is not surprising since RFC1 manifests exchange phenomena that require some conformational changes as substrates are transported into and out of cells (36). At this point, however, alterations in rates of translocation of the carrier have been reported for mutations that only occur within transmembrane domains (16, 22).
A dramatic consequence of the mutation at amino acid 45 is a striking alteration in the effects of anions on transport mediated by RFC1. In L1210 cells, a broad spectrum of inorganic and organic anions inhibit carrier-mediated folate transport, a phenomenon that appears to be charge-related (24-26). The substitution of the negatively charged glutamate with the positively charged lysine changed the nature of this interaction; in cells transfected with the mutated carrier, inorganic monovalent anions were required for carrier function. Anions also produced differences in the influx kinetic parameters for 5-CHO-THF in L1210 and MTXrA-E45K cells. In wild type cells, removal of inorganic anions increased influx by decreasing the influx Kt without a change in Vmax. This is consistent with competition at the same folate-binding site that is probably charge-related (24-26). In contrast, in cells that express the mutated carrier, inorganic anions increased the Vmax, but this was also accompanied by a small decrease in the influx Kt.
The data suggest that the conversion of a negative to a positive charge at amino acid 45 locks the carrier in an immobile position in the absence of inorganic anions in the transport medium. This might be due to attraction of the lysine to a negatively charged site, or repulsion by another positively charged residue, elsewhere in the carrier. According to this paradigm, small anions within an aqueous pocket neutralize the charge on lysine thereby restoring carrier mobility and substrate translocation. As this occurs, and transport is restored, all anions (mono- and polyanions) also interact with, and inhibit, binding of folates at a different site that accounts for anion inhibition in wild type cells, hence the parallel increase in influx Kt in the presence of anions in the MTXrA-E45K transfectant. The capacity to restore carrier function was limited to the inorganic anions studied. Neither sulfate, phosphate, nor ATP stimulated influx of 5-CHO-THF at any concentration studied, a phenomenon that may be related to their larger bulk and poor access to the lysine residue. Their somewhat weaker inhibitory effects on 5-CHO-THF influx in MTXrA-E45K as compared with L1210 cells may be due to a decreased affinity of these anions for their usual inhibitory site in the mutated RFC1.
Glutamate 45 is one of only two glutamic acid residues located in the predicted transmembrane spanning domains in murine RFC1(10). Glutamate 699 in the band 3 protein has been proposed to form a hydrogen bond with histidine 752 which is essential for band 3-mediated chloride transport (37). Hence it is possible that the RFC1 phenotype observed in MTXrA-E45K cells might be related to a loss of interaction between glutamate 45 and the histidine 289 residues, the only conserved histidine in the transmembrane domains. The specific role of the glutamate moiety will be further clarified by ongoing studies employing site-directed mutagenesis at this site.
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FOOTNOTES |
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* This work was supported by Grant CA-39807 from the National Cancer Institute.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.
Present address: Dept. of Biology, The Technion, Israel Institute
of Technology, Haifa 32000, Israel.
§ To whom requests for reprints should be addressed: Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Chanin Two, 1300 Morris Park Ave., Bronx, NY 10461.
1 The abbreviations used are: RFC1, reduced folate carrier; MTX, methotrexate; 5-CHO-THF, 5-formyltetrahydrofolate; 5-CH3THF, 5-methyltetrahydrofolate; TMQ, Trimetrexate; HBS, HEPES-buffered saline; PCR, polymerase chain reaction; kb, kilobase pair(s).
2 Richard G. Moran, personal communication.
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REFERENCES |
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