![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Graduate Program in Cancer Biology (Y.D., L.H.M.) and Department of Pharmacology (L.H.M.), Wayne State University School of Medicine, Detroit, Michigan; Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan (Z.H., C.C., J.W., L.H.M.); and Division of Medicinal Chemistry, Graduate School of Pharmaceutical Science, Duquesne University, Pittsburgh, Pennsylvania (L.W., A.G.)
Received for publication November 2, 2007.
Accepted for publication January 8, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
- and 
-carboxylates (1), hydrogen- or methyl-substituted -(2,3) or -(4,5) carboxylates, or substitutions of both - and -carboxylates (6-9) were used to inhibit [3H]MTX transport with RFC-null K562 cells expressing wt and K411A RFCs. For wt and K411A RFCs, inhibitory potencies were in the order 4 > 5 > 1 > 3 > 2; 6 to 9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2 > 1 > 4; inhibition was abolished for K411A RFC. These results establish that Lys411 participates in substrate binding via an ionic association with the substrate -carboxylate; however, this is not essential for transport. An unmodified -carboxylate is required for high-affinity substrate binding to RFC, whereas the -carboxyl is not essential.
). Because mammalian cells cannot synthesize folates de novo, these cofactors must be derived from exogenous dietary sources. Folates are hydrophilic anionic molecules with ionized - and -carboxyl groups at physiologic pH that do not cross biological membranes by diffusion alone. Accordingly, mammalian cells have evolved sophisticated transport systems for facilitating folate uptake. Although folate uptake can occur by folate receptors, organic anion transporters, and a proton-coupled folate transporter (Matherly and Goldman, 2003; Zhao and Goldman, 2007), the best characterized folate transporter is the ubiquitously expressed reduced folate carrier (RFC) (SLC19A1) (Matherly et al., 2007). Indeed, given its widespread tissue expression, RFC is considered the major transport system for folates in mammalian cells and tissues.
Membrane transport by RFC is also important for antitumor activities of antifolates used for cancer chemotherapy such as methotrexate (MTX), pemetrexed, and raltitrexed (Tomudex) (Jansen et al., 1999; Goldman and Zhao, 2002; Matherly et al., 2007). Losses of RFC function are common mechanisms of antifolate resistance in in vitro and in vivo models (Sirotnak et al., 1981; Schuetz et al., 1988; Gong et al., 1997; Jansen et al., 1998; Roy et al., 1998; Zhao et al., 1998a,b, 1999; Wong et al., 1999; Drori et al., 2000; Sadlish et al., 2000) and likely contribute to clinical resistance in patients with osteosarcoma (Guo et al., 1999) and B-precursor acute lymphoblastic leukemia who are treated with MTX (Ge et al., 2007). MTX has other clinical applications including treatment of autoimmune diseases and psoriasis (Giannini et al., 1992; Chládek et al., 1998).
|
). However, it is only since the cloning of RFCs from various species (Dixon et al., 1994; Williams et al., 1994; Moscow et al., 1995; Prasad et al., 1995; Williams and Flintoff, 1995; Wong et al., 1995) and the application of molecular biology approaches to engineer RFC for biochemical studies that a detailed picture of the molecular structure of this physiologically important carrier has emerged, including its membrane topology, its N-glycosylation, and identification of functionally and structurally important domains and amino acids (for review, see Matherly et al., 2007). In contrast, there is a dearth of information regarding the structural requirements of RFC substrates. Because RFC is an anion transporter, the role of the terminal glutamate in transport is of particular interest. Although modifications of the glutamate -carboxyl group in RFC substrates were tolerated (e.g., valine), including those for the antifolates ZD9331 and PT523 (Westerhof et al., 1995; Jansen, 1999), to date, no systematic study of the -versus -carboxylates in binding and transport by RFC has been reported.
Cationic amino acids (Arg, Lys) localized within the RFC TMD-spanning segments can be envisaged to directly participate in binding of anionic (anti)folate substrates. Of particular interest are Arg373 in TMD10 and Lys411 in TMD11 [numbering refers to human RFC (hRFC) sequence (Gen-Bank accession no. U19720
[GenBank]
)]. Both of these highly conserved amino acids were previously found to be important for RFC transport (Sharina et al., 2001; Sadlish et al., 2002; Witt and Matherly, 2002) and as likely candidates to participate in binding associations with ionized - and -carboxyl groups in folate substrates. An unidentified nucleophilic amino acid in TMD11 in hRFC was implicated as the major site of covalent modification by the activated carboxyl group(s) in N-hydroxysuccinimide (NHS) [3H]MTX (Witt et al., 2004; Hou et al., 2005), an established affinity inhibitor of RFC (Henderson and Zevely, 1984; Matherly et al., 1991).
In this report, we directly explore the role of Lys411 in TMD11 of hRFC in the binding and transport of anionic folate substrates and provide a structure-activity relationship of the substrate carboxylates for RFC transport. Our results establish that Lys411 participates in transport substrate binding to hRFC and is the primary site for covalent modification by NHS-MTX. Through the use of a novel series of furo[2,3-d]pyrimidine antifolates with substituted carboxyl groups on the terminal glutamate, we demonstrate that an ionizable -carboxyl group, but less so a -carboxyl group, is a critical substrate feature for high-affinity binding to hRFC. To our knowledge, this is the first report to systematically characterize molecular features of substrate binding to RFC in light of specific structural motifs in the (anti)folate molecule and particular conserved amino acids lining the membrane translocation pathway.
|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The sources of the antifolate drugs were as follows: raltitrexed and ZD9331 were obtained from AstraZeneca Pharmaceuticals (Maccesfield, Cheshire, UK); pemetrexed was from Eli Lilly & Co. (Indianapolis, IN); and PT523 was a gift of Dr. Andre Rosowsky (Dana Farber Cancer Institute, Boston, MA). Synthetic oligonucleotides were obtained from Invitrogen (Carlsbad, CA). Tissue culture reagents and supplies were purchased from assorted vendors with the exception of fetal bovine and supplemented calf sera, which were purchased from Hyclone Technologies (Logan, UT). Molecular biology reagents were from Promega (Madison, WI) or Invitrogen.
Synthesis of Furo[2,3-d]Pyrimidine Antifolates 1 to 9. Compound 10, 4-{1-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]propyl}-benzoic acid, was obtained as described previously (Gangjee et al., 2002). For compounds 2 to 5, 10 was coupled with the appropriate commercially available, modified, glutamate ester analogs with N-methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine in dimethylformamide at room temperature for 6 h to afford the desired esters in approximately 60% yield (see Fig. 1). Saponification with aqueous sodium hydroxide at room temperature followed by acidification to pH 4 in an ice bath afforded 2 to 5 in approximately 95% yield. For compounds 6 to 9, 10 was similarly coupled with the appropriate commercially available amines to give the desired products. A reaction scheme for the synthesis of analogs 1 to 9 is shown in Fig. 1. Detailed syntheses are provided in the Supplemental Materials and Methods.
Cell Culture. Transport-defective MTX-resistant HeLa cells, designated R5 (Zhao et al., 2004), were a generous gift of Dr. I. David Goldman (Bronx, New York). R5 cells were maintained in RPMI 1640 and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) in a humidified atmosphere at 37°C in the presence of 5% CO2. Transient transfections of wild-type (wt) and mutant hRFC constructs (see below) were performed with Lipofectamine Plus reagent (Invitrogen), as described previously (Hou et al., 2005, 2006). Cultures were split 24 h after transfection and assayed for transport and expression on Western blots after an additional 24 h.
The MTX transport-deficient K562 subline, designated K500E, was selected from wt K562 cells (American Type Culture Collection, Manassas, VA) and maintained in complete RPMI 1640 medium containing 10% supplemented calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µM MTX in a humidified atmosphere at 37°C in the presence of 5% CO2 (Wong et al., 1997). Mutant and wt hRFC constructs (see below) were transfected into K500E cells by electroporation (155 V, 1000-µF capacitance). After 24 h, cells were treated with G-418 (Geneticin; 1 mg/ml), and stable clones were selected by cloning in soft agar in the presence of 1 mg/ml G-418 (Wong et al., 1997). Transfected K500E cultures were maintained in complete RPMI 1640 medium with 1 mg/ml G-418.
Site-Directed Mutagenesis of hRFC. hRFC mutants were generated by site-directed mutagenesis using the QuickChange kit (Stratagene, La Jolla, CA) and a wt hRFC construct with a 5'-untranslated region from positions -1 to -49, the full-length hRFC open reading frame, and a hemagglutinin (HA) epitope at Gln587, cloned in pCDNA3 vector (Invitrogen) (Payton et al., 2007). Primers for site-directed mutagenesis were designed according to the instructions for the QuickChange kit and are summarized in Supplemental Table 1S. All mutations were confirmed by DNA sequencing at the Wayne State University DNA sequencing core.
Western Analysis of Mutant hRFC Transfectants. Plasma membranes were prepared by differential centrifugation (Matherly et al., 1991). For standard Western blotting, membrane proteins were electrophoresed on 7.5% polyacrylamide gels in the presence of SDS (Laemmli, 1970) and electroblotted onto polyvinylidene difluoride membranes (Pierce, Rockford, IL) (Matsudaira, 1989). hRFC proteins were detected with HA-specific mouse antibody (Covance, Berkeley, CA) and secondary IRDye 800-conjugated antibody (Rockland Immunochemicals, Gilbertsville, PA). Detection and densitometry of the blots were performed with the Odyssey Imaging System (LI-COR, Lincoln, NE).
Membrane Transport Assays. Uptake of [3H]MTX (0.5 µM) in transiently transfected R5 HeLa cells was measured over 2 min at 37°C in 60-mm dishes in an "anion-free" HEPES-sucrose-Mg2+ (HSM) buffer (20 mM HEPES, 235 mM sucrose, pH adjusted to 7.14 with MgO). Uptake of [3H]MTX was quenched with ice-cold Dulbecco's phosphate-buffered saline (DPBS). Cells were washed with ice-cold DPBS (three times) and solubilized with 0.5 N NaOH. [3H]MTX (1 µM) uptake into stably transfected K500E cells was measured over 180 s (wt and Lys411 mutant) in both HSM buffer and physiologic Hanks' balanced salt solution (HBSS) in a shaking water bath at 37°C, as described previously (Wong et al., 1997). For both cell line models, levels of intracellular radioactivity were expressed as picomoles per milligram of protein, calculated from direct measurements of radioactivity and protein contents of cell homogenates. Protein assays were based on the method of Lowry et al. (1951). For the stable transfected K500E cells, kinetic constants (Kt, Vmax) were calculated from Lineweaver-Burk plots for [3H]MTX, and Ki values for assorted antifolate substrates were determined from Dixon plots with [3H]MTX (1 µM).
Affinity Labeling of hRFC with NHS-MTX Ester. The preparation of unlabeled and radiolabeled NHS-MTX was performed exactly as described previously (Matherly et al., 1991; Witt et al., 2004). For treatments of R5 transfectants, NHS-MTX in 20 µl of dry DMSO was added to 60-mm dishes of R5 cells in 2 ml of HSM buffer at room temperature for 5 min. For nonradioactive NHS-MTX, the final concentration was 5 µM. After treatment with NHS-MTX, cells were washed three times with DPBS at 0°C and assayed for [3H]MTX (0.5 µM) transport (see above).
|
NHS esters of furo[2,3-d]pyrimidine antifolate analogs were prepared from the parent compounds exactly as for MTX. For treatments of the stable transfected K500E cells with the NHS-furo[2,3-d]pyrimidine antifolates, cells were treated in 2 ml of HSM buffer, as described above for the R5 cells, albeit in suspension in a shaking water bath at room temperature. Cells were washed with ice-cold DPBS and assayed for MTX (1 µM) transport in HBSS.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Matherly et al., 1991). NHS esterification activates the carboxyl groups of MTX and related compounds for electrophilic reaction with biological nucleophiles. NHS-[3H]MTX and -aminopterin, have been extensively used for covalently labeling the carrier in cultured cells (Henderson and Zevely, 1984; Schuetz et al., 1988; Matherly et al., 1991; Yang et al., 1992). Previous studies from our laboratory used NHS-[3H]MTX with hRFC TMD1-6 and TMD7-12 half-molecules to localize covalent binding of [3H]MTX (via the substrate carboxyl groups) to TMDs 11 and 12 (Witt et al., 2004; Hou et al., 2005). Because TMD11 (but not TMD12) is directly involved in substrate binding of hRFC substrates (Hou et al., 2005), we reasoned that one or more of the seven nucleophilic amino acids in TMD11 must be a direct target for covalent modification by the NHS-MTX activated ester and, by extension, inhibition of transport activity. Accordingly, we mutated each of the seven nucleophilic amino acids in TMD11 (Cys396, Asn403, Thr404, Thr408, Lys411, Thr412, Thr415) to non-nucleophilic amino acids (Ala or Val). Mutant and wt hRFC proteins were expressed in hRFC-null R5 HeLa cells, and all were found to be capable of transporting MTX (0.5 µM) within an approximately 3-fold range of activities (Fig. 2). When the R5 transfectants were treated with NHS-MTX (5 µM) then assayed for [3H]MTX transport, for six of seven hRFC mutants and wt hRFC, transport was inhibited. Statistically significant inhibitions resulting from NHS-MTX were measured that ranged from 24% (for Thr412) to 60% (for Thr415). Only the K411A hRFC mutant was completely and reproducibly inert to NHS-MTX treatment.
Because loss of RFC activity by NHS-MTX treatment is the result of a covalent modification of the carrier (Henderson and Zevely, 1984; Schuetz et al., 1988; Matherly et al., 1991; Witt et al., 2004), the lack of a transport effect on the K411A mutant suggested that Lys411 is a likely target for electrophilic attack by the activated NHS-MTX ester. To directly test this possibility, we transiently transfected R5 cells with wt and K411A hRFC constructs, then treated the cells with NHS-[3H]MTX (700 nM) so as to radiolabel the carrier. Plasma membranes were prepared and detergent-solubilized, and the solubilized radiolabeled proteins were fractionated on a 7.5% polyacrylamide gel for direct counting. Similar to previous reports (Matherly et al., 1991; Witt et al., 2004), for wt hRFC expressed in R5 cells, incorporation of radioactivity from NHS-[3H]MTX involved a broadly migrating hRFC protein centered at 
80 to 85 kDa (Fig. 3). Substitution of Lys411 by Ala dramatically and nearly completely abolished incorporation of radioactivity into hRFC, clearly establishing Lys411 as the primary target for covalent modification by NHS-MTX and implicating Lys411 as involved in the binding of (anti)folate substrates via ionic associations with the - and/or -carboxyl groups of the terminal glutamate.
|
50% of wt). Similar results were reported elsewhere for hRFCs with a smaller number of Lys411 mutants (Witt and Matherly, 2002; Hou et al., 2005).
Further Characterization of the Role of Lys411 in Substrate Binding with Diamino Furo[2,3-d]Pyrimidine Antifolates with Substituted Carboxyl Groups. Gangjee et al. (2002) previously reported the synthesis and biological activities of a dihydrofolate reductase inhibitor and RFC substrate, N-[4-[1-ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]L-glutamic acid (designated 1; Fig. 1), as a growth inhibitor of CCRF-CEM leukemia cells in culture. To further characterize the relative importance of the - and -carboxyl groups of (anti)folate substrates for binding with Lys411 of hRFC, we synthesized a series of furo[2,3-d]pyrimidine analogs with methyl- or hydrogen-substituted - (2, 3) or - (4, 5) carboxyl groups and with substitutions of both - and -carboxyl groups (6-9) (Fig. 1). We initially used these analogs as reversible inhibitors (at 10 µM) of radioactive MTX (1 µM) uptake with hRFC-null K562 (K500E) cells stably transfected with wt and K411A hRFC. Kinetic constants (Vmax and Kt) for MTX with wt and K411A hRFC are summarized in Table 1, and a Western blot of wt and K411A hRFC proteins in the transfected cells is shown in Supplemental Fig. 1S.
|
In physiologic HBSS buffer, the -substituted 4 and 5 analogs showed potent inhibitions of MTX transport for both wt and K411A hRFC (Fig. 5, A and B), whereas the parental drug, 1, and -substituted 2 and 3 analogs were weaker inhibitors. hRFC transport activity was stimulated (up to 5-fold) in the absence of competing anions (i.e., in anion-free HSM buffer) (data not shown), presumably reflecting decreased competition for anionic substrate binding by anions (e.g., Cl-) in HSM buffer. Consistent with this, the inhibitory effects on [3H]MTX uptake by all of the anionic antifolates with one or two carboxyl groups (compounds 1-5) were enhanced in HSM buffer (Fig. 5, C and D). Although the increase was greatest for 1, compounds 4 and 5 remained the most potent inhibitors. In contrast, analogs with neither -nor -carboxyl groups (6-9) were exceedingly poor inhibitors of MTX uptake in both HBSS and HSM buffers. Ki values for the antifolates 4 and 5 with wt and K411A hRFCs (in physiologic buffer) are summarized in Table 1. Results are consistent with higher affinity binding for antifolates 4 and 5 than for pemetrexed and raltitrexed and similar binding affinities to those for the classic hRFC high-affinity substrates PT523 and ZD9331.
|
- and -carboxyl groups participate in substrate binding to hRFC, it is the binding of the -carboxyl group that predominates and is indeed essential for high-affinity binding. From the results with the 1 versus 4/5 antifolates in HBSS, it appears that the -carboxyl can negatively affect -carboxyl group binding to the carrier. Finally, the presence of a cationic amino acid at position 411 is clearly not necessary for reversible binding of the furo[2,3-d]pyrimidine antifolates 1 to 5 to hRFC.
Affinity Inhibition of wt and K411A hRFC by NHS Esters of Diamino Furo[2,3-d]Pyrimidine Antifolates with Substituted Carboxyl Groups. To further explore the associations between the - and -carboxyl groups of (anti)folate substrates and Lys411, compound 1 and its - and -methyl-substituted congeners, 2, 4, and 6, were treated with NHS, using methods identical to those for preparing NHS-MTX. The activated NHS-antifolate esters (5 µM) were added to the K500E transfectants expressing wt and K411A hRFC (in HSM buffer) to determine effects on MTX transport activity resulting from covalent modification of the carrier, analogous to the experiment in Fig. 2 with NHS-MTX and transfected R5 cells. In striking contrast to the results for reversible inhibition of transport by the unmodified furo[2,3-d]pyrimidine antifolates (Fig. 5), for the NHS-activated analogs, the order of inhibition with wt hRFC was 2 > 1 > 4 with potencies ranging from 70% inhibition down to 30% inhibition (Fig. 6). Not surprisingly, there was no inhibition by analog 6, which has no carboxyl groups. For K411A hRFC, affinity inhibition of MTX transport by 1 to 6 was largely abolished.
|
Thus, for NHS antifolate activation and covalent modification of the carrier, the -carboxyl is clearly preferred, in contrast to the results for reversible binding in which the furo[2,3-d]pyrimidine antifolates bearing ionizable -carboxyl groups were more potent inhibitors than those with -carboxyl groups alone or with both - and -carboxyl groups. Because affinity labeling was nearly completely abolished for K411A for both NHS-MTX (see above) and the NHS-esters of the furo[2,3-d]pyrimidine antifolates, the -carboxyl groups of transport substrates must associate with Lys411 in TMD11 of hRFC.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
- and -carboxyl groups are ionized at physiologic pH, thus limiting diffusion of folates and classic antifolates across biological membranes. Because RFC is a transporter of organic anions, in this study, we focused on the mechanistic role of the substrate carboxyl groups in transport by hRFC. Analysis of membrane topology models and sequence homologies for RFCs from assorted species identified the highly conserved cationic residues, Arg373 in TMD10 and Lys411 in TMD11, as possibly functionally important (Matherly et al., 2007). By site-directed mutagenesis, both these residues were previously implicated as important to RFC transport (Sharina et al., 2001; Sadlish et al., 2002; Witt and Matherly, 2002) and as likely candidates to participate in binding associations with ionized - and -carboxyl groups in folate substrates.
This article further focuses on Lys411 and provides important new insights into the relationship between antifolate - and -carboxyl groups and this residue in TMD11, identified as an important substrate binding domain and component of the transmembrane translocation pathway in hRFC for anionic folate and antifolate substrates (Hou et al., 2005). As previously implied (Hou et al., 2006; Matherly et al., 2007), the present results establish that Lys411 lies in the proximity of the aqueous substrate binding pocket in hRFC, where it is subject to electrophilic attack by NHS-activated MTX ester and can participate in an interaction with (anti)folate substrate, primarily through an ionic association with the -carboxyl group. Remarkably, this interaction is apparently not essential for transport function because the -carboxyl group is not only expendable, but indeed its replacement by an uncharged hydrogen or a methyl group in a series of furo[2,3-d]pyrimidine antifolates actually enhances high-affinity reversible binding of substrate to the carrier, as long as an ionizable -carboxyl group is intact. Furthermore, Lys411 can be replaced by any of a number of amino acids of varying bulk and charge with relatively nominal effects on overall transport activity. From the apparently critical role of a cationic amino acid at position 373 (Sharina et al., 2001; Sadlish et al., 2002; Hou et al., 2006), we suggest that substrate binding involves an ionic association between the -carboxyl group of (anti)folate substrates and Arg373 in TMD10 of hRFC. Because substrate binding is partially preserved for antifolate analogs 2 and 3 with blocked - and ionizable -carboxylates, we propose that in the absence of the -carboxylate, the -carboxylate can adopt a folded conformation so as to mimic the -carboxylate.
Our studies are the first to systematically examine the structure-activity relationships for the - and -carboxyl groups of hRFC substrates. They are consistent with previous findings that replacement of glutamate by valine in ICI-198583 was well tolerated (Westerhof et al., 1995) and that ZD9331 and PT523, both of which have substitutions for the -carboxylate, are excellent substrates for hRFC (Jansen, 1999). However, comparisons with ZD9331 and PT523 are inexact in that the anionic character of the -carboxylate is at least partly preserved for these drugs because the benzoic acid in PT523 has the equivalent of a -carboxylate, and the tetrazole in ZD9331 is an isosteric anionic replacement for the -carboxylate.
Future studies will continue to focus on identification of functionally important amino acids in hRFC and key substrate-specific determinants of binding and translocation as important steps to understanding the mechanism of folate transport. Indeed, molecular insights from RFC structure-function studies should foster the design of new antifolate inhibitors that rely on RFC for cellular entry, or with substantially enhanced transport by other folate transporters over RFC, and the development of strategies for biochemically modulating the carrier that could be therapeutically exploited in the context of nutritional interventions or antifolate chemotherapy.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
A.G. and L.H.M. contributed equally to this work.
ABBREVIATIONS: RFC, reduced folate carrier; MTX, methotrexate; ZD9331, (2S)-2-{O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)-butyric acid; PT523, N-(4-amino-4-deoxypteroyl)-N
-hemiphthaloyl-L-ornithine; TMD, transmembrane domain; hRFC, human reduced folate carrier; NHS, N-hydroxysuccinimide; ZD1694, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-L-glutamic acid; wt, wild type; HA, hemagglutinin; HSM, HEPES-sucrose-Mg2+; DPBS, Dulbecco's phosphate-buffered saline; HBSS, Hanks' balanced salt solution; ICI-198583, (S)-2-[(1-{4-[(2-methyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-prop-2-ynyl-amino]-phenyl}-methanoyl)-amino]-pentanedioic acid.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Larry H. Matherly, Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Avenue, Detroit, MI 48201. E-mail: matherly{at}kci.wayne.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Dixon KH, Lanpher BC, Chiu J, Kelley K, and Cowan KH (1994) A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells. J Biol Chem 269: 17-20.
Drori S, Jansen G, Mauritz R, Peters GJ, and Assaraf YG (2000) Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. J Biol Chem 275: 30855-30863.
Fry DW, Yalowich JC, and Goldman ID (1982) Rapid formation of poly--glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cells in vitro. J Biol Chem 257: 1890-1896.
Gangjee A, Zeng Y, McGuire JJ, and Kisliuk RL (2002) Synthesis of N-[4-[1-ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid as an antifolate. J Med Chem 45: 1942-1948.[CrossRef][Medline]
Ge Y, Haska CL, LaFiura K, Devidas M, Linda SB, Liu M, Thomas R, Taub JW, and Matherly LH (2007) Prognostic role of the reduced folate carrier, the major membrane transporter for methotrexate, in childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group. Clin Cancer Res 13: 451-457.
Giannini EH, Brewer EJ, Kuzmina N, Shaikov A, Maximov A, Vorontsov I, Fink CW, Newman AJ, Cassidy JT, and Zemel LS (1992) Methotrexate in resistant juvenile rheumatoid arthritis. Results of the U.S.A.-U.S.S.R. double-blind, placebo-controlled trial. The Pediatric Rheumatology Collaborative Study Group and The Cooperative Children's Study Group. N Engl J Med 326: 1043-1049.[Abstract]
Goldman ID, Lichtenstein NS, and Oliverio VT (1968) Carrier-mediated transport of the folic acid analogue methotrexate, in the L1210 leukemia cell. J Biol Chem 243: 5007-5017.
Goldman ID and Zhao R (2002) Molecular, biochemical, and cellular pharmacology of pemetrexed. Semin Oncol 29: 3-17.[Medline]
Gong M, Yess J, Connolly T, Ivy SP, Ohnuma T, Cowan KH, and Moscow JA (1997) Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line. Blood 89: 2494-2499.
Guo W, Healey JH, Meyers PA, Ladanyi M, Huvos AG, Bertino JR, and Gorlick R (1999) Mechanisms of methotrexate resistance in osteosarcoma. Clin Cancer Res 5: 621-627.
Henderson GB and Zevely EM (1984) Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate. J Biol Chem 259: 4558-4562.
Hou Z, Stapels SE, Haska CL, and Matherly LH (2005) Localization of a substrate binding domain of the human reduced folate carrier to transmembrane domain 11 by radioaffinity labeling and cysteine-substituted accessibility methods. J Biol Chem 280: 36206-36213.
Hou Z, Ye J, Haska CL, and Matherly LH (2006) Transmembrane domains 4, 5, 7, 8, and 10 of the human reduced folate carrier are important structural or functional components of the transmembrane channel for folate substrates. J Biol Chem 281: 33588-33596.
Jansen G (1999) Receptor- and carrier-mediated transport systems for folates and antifolates: exploitation for folate chemotherapy and immunotherapy, in Anticancer Development Guide: Antifolate Drugs in Cancer Therapy (Jackman AL ed), pp 293-321, Humana Press Inc., Totowa, NJ.
Jansen G, Mauritz R, Drori S, Sprecher H, Kathmann I, Bunni M, Priest DG, Noordhuis P, Schornagel JH, Pinedo HM, et al. (1998) A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. J Biol Chem 273: 30189-30198.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.[CrossRef][Medline]
Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.
Matherly LH, Czajkowski CA, and Angeles SM (1991) Identification of a highly glycosylated methotrexate membrane carrier in K562 erythroleukemia cells up-regulated for tetrahydrofolate cofactor and methotrexate transport. Cancer Res 51: 3420-3426.
Matherly LH and Goldman ID (2003) Membrane transport of folates. Vitam Horm 66: 403-456.[Medline]
Matherly LH, Hou Z, and Deng Y (2007) Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer Metastasis Rev 26: 111-128.[CrossRef][Medline]
Matsudaira P (1989) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035-10038.
Moscow JA, Gong MK, He R, Sgagias MK, Dixon KH, Anzick SL, Meltzer PS, and Cowan KH (1995) Isolation of a gene encoding a human reduced folate carrier (RFC1) and analysis of its expression in transport-deficient, methotrexate-resistant human breast cancer cells. Cancer Res 55: 3790-3794.
Payton SG, Haska CL, Flatley RM, Ge Y, and Matherly LH (2007) Effects of 5'untranslated region diversity on the posttranscriptional regulation of the human reduced folate carrier. Biochim Biophys Acta 1769: 131-138.[Medline]
Prasad PD, Ramamoorthy S, Leibach FH, and Ganapathy V (1995) Molecular cloning of the human placental folate transporter. Biochem Biophys Res Commun 206: 681-687.[CrossRef][Medline]
Roy K, Tolner B, Chiao JH, and Sirotnak FM (1998) A single amino acid difference within the folate transporter encoded by the murine RFC-1 gene selectively alters its interaction with folate analogues: implications for intrinsic antifolate resistance and directional orientation of the transporter within the plasma membrane of tumor cells. J Biol Chem 273: 2526-2531.
Sadlish H, Murray RC, Williams FM, and Flintoff WF (2000) Mutations in the reduced-folate carrier affect protein localization and stability. Biochem J 346: 509-518.[CrossRef][Medline]
Sadlish H, Williams FM, and Flintoff WF (2002) Functional role of arginine 373 in substrate translocation by the reduced folate carrier. J Biol Chem 277: 42105-42112.
Schuetz JD, Matherly LH, Westin EH, and Goldman ID (1988) Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. J Biol Chem 263: 9840-9847.
Sharina IG, Zhao R, Wang Y, Babani S, and Goldman ID (2001) Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier. Mol Pharmacol 59: 1022-1028.
Sirotnak FM, Moccio DM, Kelleher LE, and Goutas LJ (1981) Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate resistant L1210 clonal cell lines derived in vivo. Cancer Res 41: 4442-4452.
Stokstad ELR (1990) Historical perspective on key advances in the biochemistry and physiology of folates, in Folic Acid Metabolism in Health and Disease (Picciano MF, Stokstad ELR, Gregory JF, eds), pp 1-21, Wiley-Liss, New York.
Westerhof GR, Schornagel JH, Kathmann I, Jackman AL, Rosowsky A, Forsch RA, Hynes JB, Boyle FT, Peters GJ, Pinedo HM, et al. (1995) Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes correlates of molecular-structure and biological activity. Mol Pharmacol 48: 459-471.[Abstract]
Williams FMR and Flintoff WF (1995) Isolation of a human cDNA that complements a mutant hamster cell defective in methotrexate uptake. J Biol Chem 270: 2987-2992.
Williams FMR, Murray RC, Underhill TM, and Flintoff WF (1994) Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake. J Biol Chem 269: 5810-5816.
Witt TL and Matherly LH (2002) Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis. Biochim Biophys Acta 1567: 56-62.[Medline]
Witt TL, Stapels S, and Matherly LH (2004) Restoration of transport activity by co-expression of human reduced folate carrier half molecules in transport impaired K562 cells: localization of a substrate binding domain to transmembrane domains 7-12. J Biol Chem 279: 46755-46763.
Wong SC, McQuade R, Proefke SA, Bhushan A, and Matherly LH (1997) Human K562 transfectants expressing high levels of reduced folate carrier but exhibiting low transport activity. Biochem Pharmacol 53: 199-206.[CrossRef][Medline]
Wong SC, Proefke SA, Bhushan A, and Matherly LH (1995) Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells. J Biol Chem 270: 17468-17475.
Wong SC, Zhang L, Witt TL, Proefke SA, Bhushan A, and Matherly LH (1999) Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier. J Biol Chem 274: 10388-10394.
Yang CH, Pain J, and Sirotnak FM (1992) Alteration of folate analogue transport inward after induced maturation of HL-60 leukemia cells: molecular properties of the transporter in an overproducing variant and evidence for down-regulation of its synthesis in maturating cells. J Biol Chem 267: 6628-6634.
Zhao R, Assaraf YG, and Goldman ID (1998a) A reduced carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate. J Biol Chem 273: 7873-7879.
Zhao R, Assaraf YG, and Goldman ID (1998b) A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function. J Biol Chem 273: 19065-19071.
Zhao R, Chattopadhyay S, Hanscom M, and Goldman ID (2004) Antifolate resistance in a HeLa cell line associated with impaired transport independent of the reduced folate carrier. Clin Cancer Res 10: 8735-8742.
Zhao R, Gao F, and Goldman ID (1999) Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier. Biochem Pharmacol 58: 1615-1624.[CrossRef][Medline]
Zhao R and Goldman ID (2007) The molecular identity and characterization of a proton-coupled folate transporter-PCFT: biological ramifications and impact on the activity of pemetrexed. Cancer Metastasis Rev 26: 129-139.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  Z. Hou and L. H. Matherly Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION J. Biol. Chem., January 30, 2009; 284(5): 3285 - 3293. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
