CHARACTERIZATION OF THE MULTIDRUG RESISTANCE PROTEIN EXPRESSED IN CELL CLONES STABLY TRANSFECTED WITH THE MOUSE MDR1 CDNA

  • 15 May 1989
    • journal article
    • research article
    • Vol. 49  (10) , 2729-2734
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, arbinocytosine, 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.

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