Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein.
Site-directed mutagenesis was used to investigate whether prolines in the predicted transmembrane domains play essential roles in the function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 13 prolines were changed to alanine were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutations of either Pro223 in transmembrane segment 4 or Pro866 in transmembrane segment 10, drastically reduced the ability of the mutant proteins to confer resistance to colchicine, adriamycin, or actinomycin D, whereas the capacity to confer resistance to vinblastine was retained. These results strongly suggest that residues in putative transmembrane segments 4 and 10, which are found in identical positions when homologous, presumably duplicated, halves of the transporter are aligned, play important roles in recognition of colchicine, adriamycin, and actinomycin D. They may either interact to form a single drug-binding site or form part of two equivalent, but independent, drug-binding sites. The lack of detectable effect of either mutation on vinblastine transport, however, indicates that there are differences in the requirements for binding of various substrates to P-glycoprotein. Mutation of Pro709 in transmembrane segment 7 resulted in a protein unable to confer drug resistance. A change at this position was found to induce a structural aberration, since the major protein product observed in transfected cells had an apparent molecular weight of 150,000, whereas the wild-type enzyme had an apparent molecular weight of approximately 170,000. Mutation of the other 10 prolines yielded protein products with structural and functional characteristics indistinguishable from wild-type P-glycoprotein.
Loo
T W
TW
Department of Medicine, University of Toronto, Ontario, Canada.
Clarke
D M
DM
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D003078:Colchicine / Q000378:metabolism / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004095:Dihydropyridines / Q000378:metabolism; D004317:Doxorubicin / Q000378:metabolism / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007672:Kidney Cortex / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011392:Proline; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D003062:Codon; D004095:Dihydropyridines; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014415:Tunicamycin; D003609:Dactinomycin; D014747:Vinblastine; C041130:azidopine; D004317:Doxorubicin; D011392:Proline; D003078:Colchicine