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I. David Goldman - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier.
Molecular pharmaceutics, 2017Co-Authors: Camille Alam, Richard H Finnell, I. David Goldman, Tozammel Hoque, Reina BendayanAbstract:Folates are essential for brain development and function. Folate transport in mammalian tissues is mediated by three major Folate transport systems, i.e., Reduced Folate Carrier (RFC), proton-coupled Folate transporter (PCFT), and Folate receptor alpha (FRα), known to be regulated by ligand-activated nuclear receptors, such as vitamin D receptor (VDR). Folate uptake at the choroid plexus, which requires the actions of both FRα and PCFT, is critical to cerebral Folate delivery. Inactivating FRα or PCFT mutations cause severe cerebral Folate deficiency resulting in early childhood neurodegeneration. The objective of this study was to investigate the role of RFC in Folate uptake at the level of the blood-brain barrier (BBB) and its potential regulation by VDR. We detected robust expression of RFC in different in vitro BBB model systems, particularly in immortalized cultures of human cerebral microvascular endothelial cells (hCMEC/D3) and isolated mouse brain capillaries. [3H]-methotrexate uptake by hCMEC/D3 ...
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Reduced Folate Carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells
American Journal of Physiology-cell Physiology, 2002Co-Authors: Rongbao Zhao, I. David GoldmanAbstract:Although the Reduced Folate Carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ∼40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport Folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ∼50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.
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Localization of the murine Reduced Folate Carrier as assessed by immunohistochemical analysis.
Biochimica et biophysica acta, 2001Co-Authors: Yanhua Wang, Rongbao Zhao, Robert G. Russell, I. David GoldmanAbstract:The Reduced Folate Carrier (RFC1) is a major route for the transport of Folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the Carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in Folate delivery in a variety of tissues. In particular, the localization of Carrier may elucidate the role of RFC1 in the vectorial transport of Folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the Folate receptor; and while RFC1 is expressed in small intestine, Folate receptor is not.
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Rescue of embryonic lethality in Reduced Folate Carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs.
The Journal of biological chemistry, 2001Co-Authors: Rongbao Zhao, Yanhua Wang, Robert G. Russell, Feng Gao, Laibin Liu, Burkhard Kneitz, Winfried Edelmann, I. David GoldmanAbstract:Abstract The Reduced Folate Carrier (RFC1) is an important route by which the major blood Folate, 5-methyltetrahydroFolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1 − /−embryos examined at E18.5 by supplementation of pregnantRFC1 +/− dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. TheseRFC1 − /− mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.
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Impact of the Reduced Folate Carrier on the accumulation of active thiamin metabolites in murine leukemia cells.
The Journal of biological chemistry, 2000Co-Authors: Rongbao Zhao, Yanhua Wang, Feng Gao, George A. Diaz, Bruce D. Gelb, I. David GoldmanAbstract:Abstract The thiamin transporter encoded bySLC19A2 and the Reduced Folate Carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of Folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially Reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations. (i) Efflux of intracellular TPP was increased in cells with high expression of RFC1. (ii) Methotrexate inhibits TPP influx. (iii) TPP competitively inhibits methotrexate influx. (iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin. The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this Carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated.
Larry H Matherly - One of the best experts on this subject based on the ideXlab platform.
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Development and validation of chemical features-based proton-coupled Folate transporter/activity and Reduced Folate Carrier/activity models (pharmacophores).
Journal of Molecular Graphics & Modelling, 2018Co-Authors: Khushbu Shah, Sudhir Raghavan, Larry H Matherly, Aleem GangjeeAbstract:Abstract All clinically used antiFolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antiFolates with selective transport into tumors over normal cells via the proton-coupled Folate transporter (PCFT) over the ubiquitously expressed Reduced Folate Carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these transporters which can be utilized for developing transporter-selective antiFolates.
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Identification of the minimal functional unit of the homo-oligomeric human Reduced Folate Carrier
The Journal of biological chemistry, 2009Co-Authors: Zhanjun Hou, Christina Cherian, Joseph Drews, Larry H MatherlyAbstract:The Reduced Folate Carrier (RFC) is the major transport system for Folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and Carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular Folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic Folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).
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A humanized mouse model for the Reduced Folate Carrier
Molecular genetics and metabolism, 2007Co-Authors: David Patterson, Christine Graham, Christina Cherian, Larry H MatherlyAbstract:The ubiquitously expressed Reduced Folate Carrier (RFC) or SLC19A1 is recognized to be an essential transport system for Folates in mammalian cells and tissues. In addition to its generalized role as a Folate transporter, RFC provides specialized tissue functions including absorption across intestinal/colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of Folates, and Folate transport across the blood-brain barrier. The human RFC (hRFC) gene is regulated by five major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5' untranslated regions (UTRs) are fused to a common splice acceptor region (positions -1 to -49) within the first coding exon with a common 1776bp coding sequence. The 5' non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5' UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5' UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5' UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue Folate uptake and distribution, none of which can be easily achieved in human populations.
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human Reduced Folate Carrier translation of basic biology to cancer etiology and therapy
Cancer and Metastasis Reviews, 2007Co-Authors: Larry H Matherly, Zhanjun Hou, Yijun DengAbstract:This review attempts to provide a comprehensive overview of the biology of the physiologically and pharmacologically important transport system termed the “Reduced Folate Carrier” (RFC). The ubiquitously expressed RFC has unequivocally established itself as the major transport system in mammalian cells and tissues for a group of compounds including Folate cofactors and classical antiFolate therapeutics. Loss of RFC expression or function may have potentially profound pathophysiologic consequences including cancer. For chemotherapeutic antiFolates used for cancer such as methotrexate or pemetrexed, synthesis of mutant RFCs or loss of RFC transcripts and proteins results in antiFolate resistance due to incomplete inhibition of cellular enzyme targets and insufficient substrate for polyglutamate synthesis. Since RFC was first cloned in 1994, tremendous advances have been made in understanding the complex transcriptional and posttranscriptional regulation of RFC, in identifying structurally and functionally important domains and amino acids in the RFC molecule as a prelude to establishing the mechanism of transport, and in characterizing the molecular defects in RFC associated with loss of transport in antiFolate resistant cell line models. Many of the insights gained from laboratory models of RFC portend opportunities for modulating Carrier expression in drug resistant tumors, and for designing a new generation of agents with improved transport by RFC or substantially enhanced transport by other Folate transporters over RFC. Many of the advances in the basic biology of RFC in cell line models are now being directly applied to human cancers in the clinical setting, most notably pediatric acute lymphoblastic leukemia and osteogenic sarcoma.
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restoration of high level transport activity by human Reduced Folate Carrier thtr1 thiamine transporter chimaeras role of the transmembrane domain 6 7 linker region in Reduced Folate Carrier function
Biochemical Journal, 2003Co-Authors: Teah L Witt, Larry H MatherlyAbstract: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.
Hamid M Said - One of the best experts on this subject based on the ideXlab platform.
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Intracellular Trafficking and Membrane Targeting Mechanisms of the Human Reduced Folate Carrier in Mammalian Epithelial
2015Co-Authors: Hamid M SaidAbstract:The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the Reduced Folate Carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cel-lular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and traffick-ing dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cyto-plasmic NH2- or COOH-terminal domains of the protein
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Identification of dynein light chain road block-1 as a novel interaction partner with the human Reduced Folate Carrier.
American journal of physiology. Gastrointestinal and liver physiology, 2009Co-Authors: Balasubramaniem Ashokkumar, Svetlana M. Nabokina, Thomas Y., Hamid M SaidAbstract:The Reduced Folate Carrier (RFC) is a major Folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in Folate absorption. Studies from our l...
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Role of Reduced Folate Carrier in intestinal Folate uptake
American journal of physiology. Cell physiology, 2006Co-Authors: Krishnaswamy Balamurugan, Hamid M SaidAbstract:Studies from our laboratory and others have characterized different aspects of the intestinal Folate uptake process and have shown that the Reduced Folate Carrier (RFC) is expressed in the gut and ...
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Role of Reduced Folate Carrier in intestinal Folate uptake
American Journal of Physiology-cell Physiology, 2006Co-Authors: Krishnaswamy Balamurugan, Hamid M SaidAbstract:Studies from our laboratory and others have characterized different aspects of the intestinal Folate uptake process and have shown that the Reduced Folate Carrier (RFC) is expressed in the gut and plays a role in the uptake process. Little, however, is known about the actual contribution of the RFC system toward total Folate uptake by the enterocytes. Addressing this issue in RFC knockout mice is not possible due to the embryonic lethality of the model. In this study, we describe the use of the new approach of lentivirus-mediated short hairpin RNA (shRNA) to selectively silence the endogenous RFC of the rat-derived intestinal epithelial cells (IEC-6), an established in vitro model for Folate uptake, and examined the effect of such silencing on Folate uptake. First we confirmed that the initial rate of [3H]folic acid uptake by IEC-6 cells was pH dependent with a markedly higher uptake at acidic compared with alkaline pH. We also showed that the addition of unlabeled folic acid to the incubation buffer lead...
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Intracellular trafficking and membrane targeting mechanisms of the human Reduced Folate Carrier in Mammalian epithelial cells.
The Journal of biological chemistry, 2002Co-Authors: Jonathan S Marchant, Veedamali S. Subramanian, Ian Parker, Hamid M SaidAbstract:The major pathway for cellular uptake of the watersoluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the Reduced Folate Carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH2- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.
Esteban E. Sierra - One of the best experts on this subject based on the ideXlab platform.
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Inhibitory effects of prostaglandin A1 on membrane transport of Folates mediated by both the Reduced Folate Carrier and ATP-driven exporters
Biochemical pharmacology, 1999Co-Authors: Yehuda G. Assaraf, Esteban E. Sierra, Solomon Babani, I. David GoldmanAbstract:Abstract Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A 1 (PGA 1 ) on processes that govern the transport of Folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the Reduced Folate Carrier, RFC1, and ATP-dependent exporters. PGA 1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a K i of ∼21 μM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA 1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity. In contrast, the effect of PGA 1 on folic acid transport was small (∼20% inhibition of total influx), consistent with the observation that the major portion of folic acid transport in CHO cells is mediated by a low pH mechanism distinct from RFC1. PGA 1 was also a potent inhibitor of the ATP-driven efflux of both MTX and folic acid. At a concentration of 7 μM PGA 1 , the efflux rate constants for these Folates were depressed by ∼70 and ∼50%, respectively. The net effects of PGA 1 on the bidirectional Folate fluxes translated into marked alterations in net transport. The addition of 7 μM PGA 1 to cells at steady state with 1 μM MTX produced a rapid onset of net uptake and the achievement of an ∼3-fold increase in the steady-state free MTX level as compared with untreated CHO cells. The addition of 7 μM PGA 1 to cells at steady state with l μM folic acid produced an ∼5-fold increase in the free Folate level. These studies establish PGA 1 as a potent inhibitor of both the Reduced Folate Carrier and ATP-driven Folate exporter(s). The noncompetitive nature of the inhibition of RFC1 is unique among anionic compounds, which are usually competitive inhibitors of the Carrier.
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Characterization of Folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective Reduced Folate Carrier function
Biochemical pharmacology, 1998Co-Authors: Esteban E. Sierra, I. David GoldmanAbstract:Abstract Folate influx at low pH was characterized in MTX r A cells, an L1210 mouse leukemia cell line with a functional defect in the Reduced Folate Carrier. Folic acid influx in MTX r A cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistinguishable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTX r A cells at pH 6.0 was 15-fold higher than at pH 7.5, in L1210 cells it was decreased by half. Influx of MTX, folic acid, 5-methyltetrahydroFolate and 5-formyltetrahydroFolate in MTX r A cells was increased at pH K t of 2.65 and 0.56 μM, and a V max of 0.45 and 0.083 nmol/g dry wt/min, respectively. MTX influx mediated by the low pH transporter was insensitive to the anionic composition of the transport buffer and affected minimally (∼20%) by Na + substitution. The anion transport inhibitors sulfobromophthalein, diisothiocyanatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene disulfonic acid were not effective inhibitors of the low pH route. MTX transport at low pH did not increase in MTX r A-R16 cells, an MTX r A derivative with 10-fold overexpression of the Reduced Folate Carrier (RFC) due to transfection with RFC1 cDNA. Inhibition of Reduced Folate Carrier activity with acetamidoisothiocyanatostilbene disulfonic acid resulted in identical MTX influx in L1210, MTX r A, and MTX r A-R16 cells at pH 5.5. Finally, low pH-mediated MTX influx was Reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensin ≫ valinomycin). The data indicate that L1210 and MTX r A cells express similar activities of a low pH Folate transporter that has properties distinct from, and independent of, the Reduced Folate Carrier.
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Organization of the murine Reduced Folate Carrier gene and identification of variant splice forms
Biochimica et biophysica acta, 1997Co-Authors: Kevin E. Brigle, Michael J. Spinella, Esteban E. Sierra, I. David GoldmanAbstract:Abstract RT-PCR analysis of the Reduced Folate Carrier (RFC) from L1210 and murine erythroleukemia cells led to the identification of three clones which appeared to result from the use of alternative splice sites. The nucleotide sequence of each splice form predicts a protein that contains at least the first 7 transmembrane domains of the parental RFC protein followed by a novel hydrophilic carboxyl terminus of 33, 72, or 105 amino acid residues. Sequence analysis of cDNA clones isolated from murine liver and the results of 5′-RACE from L1210 cells indicated that RFC also utilizes alternate 5′-terminal exons. To understand how the alternatively spliced RFC transcripts and multiple 5′-termini were generated, the genomic organization of RFC was determined. The gene is comprised of at least 8 exons, the first two of which encode the alternative 5′ termini. Based on sequence identity with cDNAs encoding RFC from hamster and rat, however, it appears that additional 5′ exons may be present. Two of the RFC splice variants result from the use of a cryptic splice donor site within exon 4 and the third results from the use of a cryptic splice acceptor site within exon 5. In addition, the splice variant form that encodes the largest protein also utilizes an alternative exon located between exons 5 and 6. The apparent use of alternative transcriptional start sites and the identification of several RFC splice forms raises the possibility that unique RFC molecules may be generated that exhibit tissue- or cell line-specific distribution.
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pH dependence of methotrexate transport by the Reduced Folate Carrier and the Folate receptor in L1210 leukemia cells. Further evidence for a third route mediated at low pH
Biochemical pharmacology, 1997Co-Authors: Esteban E. Sierra, Kevin E. Brigle, Michael J. Spinella, I. David GoldmanAbstract:F2-MTXrA is an L1210 leukemia cell line with a functional defect in the Reduced Folate Carrier and high level expression of Folate receptor β. The pH-dependence of methotrexate (MTX) influx by Folate receptor β in F2-MTXr A cells was characterized and compared with that of the Reduced Folate Carrier in parental L1210 cells. MTX influx by Folate receptor β had a pH optimum of 6.5, whereas influx mediated by the Reduced Folate Carrier showed a pH optimum of 7.5. Increased Folate receptor β-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTXrA cells proceeded by another mechanism. This transport route became active at pH
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ph dependence of methotrexate transport by the Reduced Folate Carrier and the Folate receptor in l1210 leukemia cells further evidence for a third route mediated at low ph
Biochemical Pharmacology, 1997Co-Authors: Esteban E. Sierra, Michael J. Spinella, Kevin E. Brigle, David I GoldmanAbstract:F2-MTXrA is an L1210 leukemia cell line with a functional defect in the Reduced Folate Carrier and high level expression of Folate receptor β. The pH-dependence of methotrexate (MTX) influx by Folate receptor β in F2-MTXr A cells was characterized and compared with that of the Reduced Folate Carrier in parental L1210 cells. MTX influx by Folate receptor β had a pH optimum of 6.5, whereas influx mediated by the Reduced Folate Carrier showed a pH optimum of 7.5. Increased Folate receptor β-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTXrA cells proceeded by another mechanism. This transport route became active at pH <7.5, operated optimally at pH 6.0 to 6.5, and, unlike Folate receptor β-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a Ki of 5.3 μM and a Vmax of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a Ki of 148 μM, and had similar relative affinities for folic acid, leucovorin, and 5-methyltetrahydroFolate. Influx of 5-methyltetrahydroFolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTXrA cells, optimal at low pH and distinct from the Reduced Folate Carrier or the Folate receptor.
Teah L Witt - One of the best experts on this subject based on the ideXlab platform.
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restoration of high level transport activity by human Reduced Folate Carrier thtr1 thiamine transporter chimaeras role of the transmembrane domain 6 7 linker region in Reduced Folate Carrier function
Biochemical Journal, 2003Co-Authors: Teah L Witt, Larry H MatherlyAbstract: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.
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Restoration of high-level transport activity by human Reduced Folate Carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in Reduced Folate Carrier function.
The Biochemical journal, 2003Co-Authors: Teah L Witt, Larry H MatherlyAbstract: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.
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Repression of Human Reduced Folate Carrier Gene Expression by Wild Type p53
The Journal of biological chemistry, 2000Co-Authors: Bee Ching Ding, Teah L Witt, Johnathan R. Whetstine, John D. Schuetz, Larry H MatherlyAbstract:Abstract The relationship between loss of functional p53 and human Reduced Folate Carrier (hRFC) levels and function was examined in REH lymphoblastic leukemia cells, which express wild type p53, and in p53-null K562 cells (K562pTet-on/p53) engineered to express wild type p53 under control of a tetracycline-inducible promoter. Activation of p53 in REH cells by treatment with daunorubicin was accompanied by decreased (∼5-fold) levels of hRFC transcripts and methotrexate transport. Treatment of K562pTet-on/p53 cells with doxycycline resulted in a dose-dependent expression of p53 protein and transcripts, increased p21 protein, decreased dihydroFolate reductase, and G1 arrest with decreased numbers of cells in S-phase. p53 induction was accompanied by up to 3-fold decreases in hRFC transcripts transcribed from the upstream hRFC-B promoter and similar losses of hRFC protein and methotrexate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and dihydroFolate reductase without effects on hRFC levels or activity. When the hRFC-B promoter was expressed as full-length and basal promoter-luciferase reporter constructs in K562pTet-on/p53 cells, induction of p53 with doxycycline resulted in a 3-fold loss of promoter activity, which was reversed by cotransfection with a trans-dominant-negative p53. These studies show that wild type p53 acts as a repressor of hRFC gene expression, via a mechanism that is independent of its effects on cell cycle progression.