The minimum functional unit of human P-glycoprotein appears to be a monomer.
Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His)10, even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drug-stimulated ATPase activity when the half-molecules NH2-terminal half-(His)10/COOH-terminal half-A52 or NH2-terminal half-A52/COOH-terminal half-(His)10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer.
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Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario, Canada M5S 1A8.
Clarke
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D018432:Drug Resistance, Multiple; D006801:Humans; D007668:Kidney; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins