Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein

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Abstract
A radioiodinated photoaffinity analogue of methotrexate, N.alpha.-(4-amino-4-deoxy-10-methylpteroyl)-N.epsilon.-(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., and Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (21210/R81). from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37.degree.C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37.degree.C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. The protoprobe eventually dissociated from the 38K protein and was found associated with other intracellular proteins at 10-12 min, corresponding to the time required for methotrexate uptake to reach a steady-state level. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. However, the uptake of radiolabeled APA-ASA-Lys by L1210 cells was inhibited by methotrexate but not by folate, suggesting the binding/transport proteins for folate and reduced folate coenzymes are separate but may be immunologically related. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets.