ISOLATION OF HUMAN CDNAS THAT RESTORE METHOTREXATE SENSITIVITY AND REDUCED FOLATE CARRIER ACTIVITY IN METHOTREXATE TRANSPORT-DEFECTIVE CHINESE-HAMSTER OVARY CELLS

This report describes the isolation, nucleotide sequencing, and functi onal expression of human cDNAs that restore reduced folate carrier act ivity in transport-defective cells. Based on homology to a partial mur ine cDNA probe, two functional cDNAs were isolated from a lambda gt11 library prepared from methotrexate transport upregulated K562 cells (K 562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a 1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an internal dele tion (626 base pairs) resulting a shortened open reading frame and 3'- untranslated region. KS43 and KS32 encoded proteins with multiple hydr ophobic domains, one consensus N-glycosylation site, and predicted mol ecular masses of 65 and 58 kDa, respectively. The deduced amino acid s equence of KS43 is 79% and 80% homologous to the mouse and hamster seq uences, respectively (Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, R., 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. Bi ol. Chem. 269, 5810-5816). Northern blots identified one primary trans cript at 3.1 kb in parental K562, R562.4CF, and transport-impaired K50 0E cells; transcript levels varied by 7-fold. The expression of both K S43 and KS32 in methotrexate transport-defective Chinese hamster ovary cells restored methotrexate sensitivity and transport. Certain transp ort characteristics of the transfected cells resembled both the wild t ype human (K562) and hamster ''classical'' reduced folate carriers, su ggesting the expression of a hybrid system. For instance, based on K-i values, up to a 4-fold increased affinity for 1843U89 over wild type hamster cells (typical of human cells), and a 19-fold increased affini ty for methotrexate over K562 cells (typical of hamster cells) was obs erved. Farther, a photoaffinity probe with high specificity for the re duced folate carrier labeled 94-kDa proteins in K562 cells and the tra nsfectant containing the full length KS43, and a 85-kDa protein in the transfectant containing the 3'-truncated KS32. No specifically labele d proteins were de tected in wild type or mock-transfected hamster cel ls. Collectively, our results suggest that the KS43/KS32 cDNAs encode the human reduced folate carrier; however, additional modulatory/regul atory factors may be required to manifest the full spectrum of transpo rt substrate activities typical of this system.