Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells.
A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM). The parent compounds of the iodinated photoprobe inhibit [3H]methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM. UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.
Price
E M
EM
Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, Ohio 45267-0522.
Freisheim
J H
JH
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins; D013329:Structure-Activity Relationship
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; C489021:methotrexate transporter protein, mouse; C053282:N(alpha)-(4-amino-4-deoxy-10-methylpteroyl)-N(epsilon)-(4-azido-5-iodosalicylyl)lysine; D008727:Methotrexate