Abstract
Structural features of the multidrug resistance protein encoded by the mouse mdr1 gene were studied in multidrug-resistant cell clones stably transfected with a biologically active cDNA clone. Independently derived transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, mitoxantrone, VP-16, and vinblastine but remained sensitive to cis-platinum, 5-fluorouracil, arabinocytosine, and bleomycin. In drug-resistant transfectants the mdr1 gene product was greatly overexpressed as a polypeptide of apparent molecular weight 160,000–170,000. This protein was present in membrane enriched fractions and could be metabolically labeled with [3H]glucosamine, confirming that the transfected mdr1 gene encodes a membrane glycoprotein. The protein was found phosphorylated on serine residues and was shown to be photolabeled by both the calcium antagonist azidopine and the ATP analogue 8-azido ATP. Tryptic mapping of the ATP-photoaffinity labeled protein indicated that ATP cross-linking was site-specific and limited to two discrete peptide fragments of the protein, suggesting that the overexpressed mdr protein is capable of direct and specific ATP binding.
Footnotes
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↵1 Supported by operating grants from the Medical Research Council (MRC), the National Cancer Institute of Canada, and the Fonds de Recherche en Santé du Québec (FRSQ).
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↵2 Recipient of a post-doctoral fellowship from the Deutscher Akademischer Austauschdienst (DAAD), Sonderprogramm “Molekulare Parasitologie.”
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↵3 Recipient of a studentship from National Science and Engineering Research Council of Canada.
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↵4 Recipient of an MRC scholarship.
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↵5 To whom requests for reprints should be addressed, Department of Biochemistry, McGill University, 3655 Drummond, Room 902, Montreal, Quebec H3G 1Y6, Canada.
- Received May 11, 1988.
- Revision received December 12, 1988.
- Accepted February 17, 1989.
- ©1989 American Association for Cancer Research.
Volume 49, Issue 10
