Send to

Choose Destination
See comment in PubMed Commons below
J Biol Chem. 1995 Jul 21;270(29):17468-75.

Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells.

Author information

  • 1Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201, USA.


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 lambda gt11 library prepared from methotrexate transport upregulated 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. 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 Ki 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.

[PubMed - indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Support Center