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

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A phenylalanine substitution for serine in the reduced folate carrier at residue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cell line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folate source. The transport characteristics of the mutated carrier were studied by transfection into the MTX(r)A line, which lacks endogenous RFC1 function. The level of expression of carrier in the cell lines studied was determined by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF). Influx of 5-CH3-THF and 5-CHO-THF mediated by RFC1-S309F was 20- and 7-fold greater than that of MTX, respectively. Consistent with the influx difference between 5-CHO-THF and MTX, the growth requirement (EC50) for 5-CHO-THF in MTX(r)A-S309F cells was decreased by a factor of 9, while the MTX IC50 was reduced by a factor of only ~2 as compared with the recipient MTX(r)A cells. The decrease in 5-CH3-THF influx mediated by the mutated carrier was attributed to a decrease in the mobility of the 5-CH3-THF-carrier complex, since the influx K(t) was essentially unchanged. However, the reduction in 5-CHO-THF and MTX influx was attributed to decreases in both carrier affinity and V(max), although the decline in the MTX influx V(max) appeared to be much greater than for 5-CHO-THF. The inhibitory effect of chloride on 5-CHO-THF influx observed for L1210 cells was eliminated in the MTX(r)A-S309F line. This study represents another example of a single mutation in RFC1 that markedly impairs MTX influx but partially preserves transport of reduced folates when cells are selected with 5-CHO-THF as the available folate substrate. The data indicate that residues in the predicted eighth transmembrane domain of RFC1 can play an important role in the selectivity of folate binding and the mobility of the carrier-substrate complex. Copyright (C) 1999 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)1615-1624
Number of pages10
JournalBiochemical Pharmacology
Issue number10
Publication statusPublished - Nov 15 1999



  • Antifolates
  • Carrier-mediated transport
  • Folates
  • Major Facilitator Superfamily
  • Reduced folate carrier
  • anticancer drug resistance

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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