Identification of a highly glycosylated methotrexate membrane carrier in K562 human erythroleukemia cells up-regulated for tetrahydrofolate cofactor and methotrexate transport.
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 Vmax. 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 Mr 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-beta-galactosidase, reduced the apparent molecular weight to Mr approximately 52,000 or approximately 58,000, respectively. These results suggest that the high-affinity transporter in K562.4CF cells is an N-linked glycoprotein containing internal beta-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.
Matherly
L H
LH
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
Czajkowski
C A
CA
Angeles
S M
SM
D000345:Affinity Labels; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D002955:Leucovorin; D008727:Methotrexate