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 Journal Article


Authors: Roy, K.; Tolner, B.; Chiao, J. H.; Sirotnak, F. M.
Article Title: 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
Abstract: The apparent K(m), but not V(max), for influx of methotrexate (MTX) mediated through the plasma membrane of S180 cells by the one-carbon, reduced folate transporter as well as the K(D) for binding to the transporter were 4- fold higher than in L1210 cells correlating with the greater intrinsic resistance of the former to this folate analogue. In contrast, no difference was observed between each cell type with regard to efflux of [3H]MTX mediated by this same transporter in ATP-depleted cells. The difference in influx K(m) in the case of this 10-methyl substituted N1O analogue of folic acid was not seen with more effective permeants, such as the unsubstituted N1O aminopterin or C1O analogues. Thus, values for influx K(m) for aminopterin, which were 1-1.2 μM in each cell type, increased as a result of substitution at N1O (MTX) 3-fold in L1210 cells but 12-fold in S180 cells. Nucleotide sequencing of reverse transcriptase-polymerase chain reaction- generated cDNA and of polymerase chain reaction-generated genomic DNA identified a single nucleotide difference between each cell type at +890 within exon 3 of the RFC-1 gene. This was in the form of a G (L1210 cells) to A (S180 cells) transition. Codon 297, the site of this transition, encodes either Ser or Asn in L1210 or S180 cells, respectively, which is located between the seventh and eight membrane-spanning helices. This amino acid difference had no effect on the electrophoretic mobility or amount of the transporter in each cell type that was shown by Western blotting with anti- RFC-1 peptide antibodies to migrate as 46 kDa in each case. Proof that this nucleotide difference alone accounted for the alteration in influx between each cell type was obtained by S180 RFC-1 cDNA versus L1210 RFC-1 cDNA transfection of an L1210 cell variant with undetectable MTX influx and RFC-1 gene expression. In this case, the higher K(m) for MTX influx associated with S180 cells was duplicated only in the S180 RFC-1 transfectants. These results appear to document the first example of a nucleotide alteration within the RFC-1 gene, which influences the interaction of MTX with the encoded plasma membrane transporter. An analysis of topology, in addition to other considerations, suggests that the site of the amino acid difference found in the transporter from L1210 and S180 cells occurs within or near the binding site on the external plasma membrane surface.
Keywords: nonhuman; methotrexate; protein conformation; animal cell; mouse; animals; mice; reverse transcription polymerase chain reaction; antimetabolites, antineoplastic; neoplasm proteins; membrane proteins; drug resistance, neoplasm; tumor cells, cultured; cell type; amino acid sequence; molecular sequence data; sequence homology, amino acid; nucleotide sequence; carrier proteins; cell membrane; binding site; models, molecular; protein folding; folic acid antagonists; membrane transport; biological transport; aminopterin; internalization; electrophoretic mobility; amino acid transport; membrane transport proteins; amino acid metabolism; leukemia l1210; potassium transport; folic acid derivative; priority journal; article; sarcoma 180
Journal Title: Journal of Biological Chemistry
Volume: 273
Issue: 5
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1998-01-30
Start Page: 2526
End Page: 2531
Language: English
DOI: 10.1074/jbc.273.5.2526
PUBMED: 9446553
PROVIDER: scopus
DOI/URL:
Notes: Article -- Export Date: 12 December 2016 -- Source: Scopus
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MSK Authors
  1. Francis M Sirotnak
    122 Sirotnak
  2. Krishnendu K Roy
    20 Roy