A SINGLE AMINO-ACID DIFFERENCE WITHIN THE FOLATE TRANSPORTER ENCODED BY THE MURINE RFC-1 GENE SELECTIVELY ALTERS ITS INTERACTION WITH FOLATE ANALOGS - IMPLICATIONS FOR INTRINSIC ANTIFOLATE RESISTANCE AND DIRECTIONAL ORIENTATION OF THE TRANSPORTER WITHIN THE PLASMA-MEMBRANE OF TUMOR-CELLS

The apparent K-m, but not V-max, for influx of methotrexate (MTX) medi ated through the plasma membrane of S180 cells by the one-carbon, redu ced folate transporter as well as the K-D for binding to the transport er were 4-fold higher than in L1210 cells correlating with the greater intrinsic resistance of the former to this folate analogue, In contra st, no difference was observed between each cell type with regard to e fflux of [H-3]MTX mediated by this same transporter in ATP-depleted ce lls, The difference in influx K-m in the case of this 10-methyl substi tuted N10 analogue of folic acid was not seen with more effective perm eants, such as the unsubstituted N10 aminopterin or C10 analogues, Thu s, values for influx K-m, for aminopterin, which were 1-1.2 mu M in ea ch cell type, increased as a result of substitution at NIO (MTX) 3-fol d in L1210 cells but 12-fold in S180 cells. Nucleotide sequencing of r everse transcriptase-polymerase chain reaction-generated cDNA and of p olymerase chain reaction-generated genomic DNA identified a single nuc leotide 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 cell s) transition, Codon 297, the site of this transition, encodes either Ser or Asn in L1210 or S180 cells, respectively, which is located betw een the seventh and eight membrane-spanning helices, This amino acid d ifference had no effect on the electrophoretic mobility or amount of t he transporter in each cell type that was shown by Western blotting wi th anti-RFC-1 peptide antibodies to migrate as 46 kDa in each case, Pr oof that this nucleotide difference alone accounted for the alteration in influx between each cell type was obtained by S180 RFC-1 cDNA vers us L1210 RFC-1 cDNA transfection of an L1210 cell variant with undetec table 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 th e S180 RFC-1 transfectants. These results appear to document the first example of a nucleotide alteration within the RFC-1 gene, which influ ences the interaction of MTX with the encoded plasma membrane transpor ter, An analysis of topology, in addition to other considerations, sug gests that the site of the amino acid difference found in the transpor ter from L1210 and S180 cells occurs within or near the binding site o n the external plasma membrane surface.