title: Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides.
abstract: P-glycoprotein consists of two homologous halves, each composed of six potential transmembrane sequences and an ATP-binding domain. The cDNA coding for human P-glycoprotein was divided in half and subcloned into separate plasmids in order to express each half as a separate polypeptide and to characterize its contribution to function. Expression of cDNAs coding for either the NH2- or COOH-terminal half-molecules in HEK 293 cells yielded products of 88 and 64 kDa, respectively. The NH2-terminal half-molecule was glycosylated, since its size decreased from 88 to 79 kDa when expressed in the presence of tunicamycin. No change was observed in the size of the COOH-terminal half-molecule when it was expressed in the presence of tunicamycin, indicating that it was not glycosylated. The cDNAs coding for each half of P-glycoprotein were transfected into NIH-3T3 cells to test for biological activity. No drug-resistant colonies were obtained when cells were transfected with cDNA coding for each half-molecule or when cells were co-transfected with both cDNAs, although stable expression of each half-molecule was detected. The inability to confer drug resistance was likely due to a defect in targeting of the half-molecules to the cell surface. Each half-molecule was then expressed in Sf9 insect cells using a baculovirus vector to allow measurement of partial function. The half-molecules exhibited ATPase activity, but their activities were not stimulated by drug substrates. Drug-stimulatable ATPase activity was present, however, when the half-molecules were expressed together. These results suggest that coupling of ATPase activity to drug binding requires interaction between both halves of P-glycoprotein.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: T W
authors.initials: TW
authors.lastname: Loo
authors.forename: D M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary; D004351:Drug Resistance; D004789:Enzyme Activation / Q000187:drug effects; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D000251:Adenosine Triphosphatases