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(Received for publication, January
18, 1995; and in revised form, April 7, 1995) From the This report describes the isolation, nucleotide sequencing, and
functional expression of human cDNAs that restore reduced folate
carrier activity in transport-defective cells. Based on homology to a
partial murine cDNA probe, two functional cDNAs were isolated from a
The expression of both KS43 and
KS32 in methotrexate transport-defective Chinese hamster ovary cells
restored methotrexate sensitivity and transport. Certain transport
characteristics of the transfected cells resembled both the wild type
human (K562) and hamster ``classical'' reduced folate
carriers, suggesting the expression of a hybrid system. For instance,
based on K
Volume 270,
Number 29,
Issue of July 21, pp. 17468-17475, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
gt11 library prepared from methotrexate transport up-regulated
K562 cells (K562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a
1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an
internal deletion (626 base pairs) resulting a shortened open reading
frame and 3`-untranslated region. KS43 and KS32 encoded proteins with
multiple hydrophobic domains, one consensus N-glycosylation
site, and predicted molecular masses of 65 and 58 kDa, respectively.
The deduced amino acid sequence of KS43 is 79% and 80% homologous to
the mouse and hamster sequences, respectively (Dixon, K. H., Lanpher,
B. C., Chiu, J., Kelley, K., and Cowan, K. H.(1994) J. Biol. Chem. 269, 17-20; Williams, F. M. R., Murray, R. C., Underhill, T.
M., and Flintoff, W. F.(1994) J. Biol. Chem. 269,
5810-5816). Northern blots identified one primary transcript at
3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells;
transcript levels varied by 7-fold.
values, up to a 4-fold
increased affinity for 1843U89 over wild type hamster cells (typical of
human cells), and a 19-fold increased affinity for methotrexate over
K562 cells (typical of hamster cells) was observed. Further, a
photoaffinity probe with high specificity for the reduced folate
carrier labeled 94-kDa proteins in K562 cells and the transfectant
containing the full-length KS43, and a 85-kDa protein in the
transfectant containing the 3`-truncated KS32. No specifically labeled
proteins were detected in wild type or mock-transfected hamster cells.
Collectively, our results suggest that the KS43/KS32 cDNAs encode the
human reduced folate carrier; however, additional modulatory/regulatory
factors may be required to manifest the full spectrum of transport
substrate activities typical of this system.
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