Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA.
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 crosslinking 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.
Schurr
E
E
Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Canada.
Raymond
M
M
Bell
J C
JC
Gros
P
P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D004247:DNA / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000235:genetics; D010766:Phosphorylation; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D008562:Membrane Glycoproteins; D000255:Adenosine Triphosphate; D004247:DNA