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J Biol Chem, Vol. 273, Issue 30, 19065-19071, July 24, 1998
From the Departments of Medicine and Molecular
Pharmacology, and the Albert Einstein Comprehensive Cancer Center,
Albert Einstein College of Medicine, Bronx, New York 10461
In an ongoing study of
structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified
in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and
5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The
characteristics of the mutated carrier, RFC1-E45K, were studied by
cDNA transfection into the murine MTXrA line in
which endogenous carrier is not functional. Folic acid influx doubled
in the transfectant MTXrA-E45K as compared with L1210 or
MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only
14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in
MTXrA-E45K cells was characterized by a 12- and 3.6-fold
decrease in influx Vmax and
Kt respectively, relative to L1210 cells. The folic
acid influx Ki in L1210 cells was more than 50-fold
greater than that of MTX based upon inhibition of 5-CHO-THF influx. In
comparison, the mutated carrier had comparable affinities for folic
acid and MTX in MTXrA-E45K cells due to a 7-fold decrease
in the folic acid influx Ki and 7-fold increase in
the MTX influx Ki. Transport via native RFC1 is
inhibited by a variety of anions in L1210 cells associated with an
increase in influx Kt. However, influx of 5-CHO-THF
in MTXrA-E45K cells in a HEPES buffer (9 mM
chloride) was decreased by 70% due to a 3-fold fall in the
Vmax. In the complete absence of chloride
(K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10%
that in HBS buffer. 5-CHO-THF influx was restored by addition of
chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP
which were all inhibitory over a broad range of
concentrations.
The data suggest that substitution of a positive for a
negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when
cells are placed under selective pressure with antifolates that utilize
RFC1 as the major route of entry into mammalian cells.
A Mutated Murine Reduced Folate Carrier (RFC1) with Increased
Affinity for Folic Acid, Decreased Affinity for Methotrexate, and an
Obligatory Anion Requirement for Transport Function
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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