The basis for impaired reduced folate carrier (RFC) activity in methotrexate-resistant CCRF-CEM (CEM/ Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3.1-kilobase RFC transcript. A ϳ85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/ Mtx-1 transcripts at positions 130 (P 1 ; changes glycine 44 3 arginine) and 380 (P 2 ; changes serine 127 3 asparagine Despite the availability of newer antifolates, methotrexate (Mtx) 1 continues to play an important role as an antineoplastic agent. To reach its intracellular target, dihydrofolate reductase, the preferred route of Mtx entry involves the reduced folate carrier (RFC; 1, 2). RFC transport of Mtx is critical to drug action because of its role in generating sufficient unbound intracellular antifolate to sustain maximal enzyme inhibition (1). Furthermore, high levels of Mtx are also necessary for the synthesis of Mtx polyglutamates (1).Defective membrane transport of Mtx by RFC has been identified as a major mechanism of Mtx resistance (1-11). Transport alterations can manifest as reduced rates of carrier translocation (reduced V max ), decreased affinities for transport substrates (increased K t ), or both, and may involve decreased levels of normal RFC (6) or the expression of structurally altered RFC proteins (7-11). For instance, in Mtx-resistant K562 (K500E) cells, impaired Mtx transport is accompanied by decreased RFC transcripts and protein (6). A G to A transition at position 890 of the murine RFC cDNA resulted in a substitution of serine 297 by asparagine and a selective decrease in Mtx binding affinity (ϳ4-fold) without effects on other antifolate analogs (aminopterin, Ref. 9). Likewise, replacement of serine 46 by asparagine (10) or glutamate 45 by lysine (11) in murine RFC resulted in greater impairment of uptake for Mtx than (6S)-5-formyl tetrahydrofolate. In severely transport defective L1210 cells (Mtx r A), loss of transport activity appeared to reflect a single (G to C) point mutation at nucleotide 429 of the murine RFC cDNA sequence which resulted in the substitution of proline 130 by alanine (7). However, these cells also contained a wild-type RFC allele that was not transcribed. A silent wild-type RFC allele was described for Mtx-resistant MOLT-3 cells (MOLT-3/Mtx 10,000 ; Ref. 8). Moreover, two mutations in the RFC coding region were detected which resulted in the creation of new stop codons and synthesis of truncated nonfunctional RFCs (8).In this report, the molecular mechanisms responsible for the transport-impaired phenotype (ϳ3% of wild-type) of Mtx-resistant (ϳ243-fold) CCRF-CEM (CEM/Mtx-1;12) cells were examined. We show that although the levels of RFC transcripts are essentially unchanged from wild-type cells, there is a complete loss of RFC protein due to ear...
Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49 -61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.
Reduced folates such as 5-methyl tetrahydrofolate and classical antifolates such as methotrexate are actively transported into mammalian cells by the reduced folate carrier (RFC). RFC is characterized by 12 stretches of mostly hydrophobic, ␣-helix-promoting amino acids, internally oriented N and C termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. Previous studies showed that deletion of the majority of the central loop domain between TMDs 6 and 7 abolished transport, but this segment could be replaced with mostly non-homologous sequence from the SLC19A2 thiamine transporter to restore transport function. In this report, we expressed RFC from separate TMD1-6 and TMD7-12 RFC halfmolecule constructs, each with a unique epitope tag, in RFC-null K562 cells to restore transport activity. Restored transport exhibited characteristic transport kinetics for methotrexate, a capacity for trans-stimulation by pretreatment with leucovorin, and inhibition by Nhydroxysuccinimide methotrexate, a documented affinity inhibitor of RFC. The TMD1-6 half-molecule migrated on SDS gels as a 38 -58 kDa glycosylated species and was converted to 27 kDa by N-glycosidase F or tunicamycin treatments. The 40 kDa TMD7-12 half-molecule was unaffected by these treatments. Using transfected cells expressing both TMDs 1-6 and TMDs 7-12 as separate polypeptides, the TMD7-12 half-molecule was covalently radiolabeled with N-hydroxysuccinimide [ 3 H]methotrexate. No radioactivity was incorporated into the TMD1-6 half-molecule. Digestion with endoproteinase GluC decreased the size of the radiolabeled 40 kDa TMD7-12 polypeptide to ϳ20 kDa. Our results demonstrate that a functional RFC can be reconstituted with RFC half-molecules and localize a critical substrate binding domain to within TMDs 7-12.
The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1-6 and TMDs 7-12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204-214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFC(D215-R263 Delta), hRFC(K204-R263 Delta) and hRFC(K204-R214 Delta) proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6 S )5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFC(K204-R263 Delta) restored low levels of transport (16-21% of the wild type). Insertion of the ThTr1 linkers into hRFC(D215-R263 Delta) at position 215 restored 60-80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.