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Identification of a Highly Glycosylated Methotrexate Membrane Carrier in K562 Human Erythroleukemia Cells Up-regulated for Tetrahydrofolate Cofactor and Methotrexate Transport¹

Development Therapeutics Program, Michigan Cancer Foundation, Detroit, Michigan 48201

A K562 human erythroleukemia line (designated K562.4CF) was selected for increased tetrahydrofolate cofactor transport in a growth-limiting concentration (0.4 nM) of (6R,S)-5-formyltetrahydrofolate. K562.4CF cells exhibited elevated methotrexate uptake relative to parental cells, attributable to a 10-fold increased influx V_max. The rate of methotrexate efflux in K562.4CF cells was somewhat increased (55%) as well. The transport system in K562.4CF cells had similar and high apparent binding affinities for methotrexate and 5-formyltetrahydrofolate and a markedly reduced affinity for folic acid, properties typically associated with the "classical" methotrexate/tetrahydrofolate cofactor transporter in tumor cells. Methotrexate uptake in K562.4CF cells decreased substantially under nonselective conditions; high levels of transport were restored in 0.4 nM 5-formyltetrahydrofolate. Treatment of parental and K562.4CF cells with N-hydroxysuccinimide methotrexate inhibited methotrexate influx. N-Hydroxysuccinimide-[³H]methotrexate (700 nM) radiolabeled a broadly migrating band at M_r 76,000–85,000. Incorporation from N-hydroxysuccinimide-[³H]methotrexate into this band was increased 7-fold in K562.4CF over parental cells and was blocked by unlabeled methotrexate, (6S)-5-formyltetrahydrofolate, or, to a lesser extent, folic acid. Whereas incubation with endoglycosidase F had no effect on the electrophoretic migration of the labeled protein, treatment with endoglycosidase F and glycopeptidase F, or endo-ß-galactosidase, reduced the apparent molecular weight to M_r 52,000 or 58,000, respectively. These results suggest that the high-affinity transporter in K562.4CF cells is an N-linked glycoprotein containing internal ß-galactosidic linkages in, or immediately after, unbranched poly-N-acetyllactosamine sequences. Differences in the level of glycosylation may, in part, account for the disparity in the apparent sizes of the homologous folate transport proteins from human and murine cells.

¹ Supported by grants from the American Cancer Society (CH 435), the Children's Leukemia Foundation of Michigan, and the Elsa U. Pardee Foundation.

² To whom requests for reprints should be addressed, at Michigan Cancer Foundation, 110 East Warren Avenue, Detroit, MI 48201.

Received 12/11/90. Accepted 4/17/91.

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