title: The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.
abstract: The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.
authors.affiliation: Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: T W
authors.initials: TW
authors.lastname: Loo
authors.forename: D M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002211:Capsaicin / Q000494:pharmacology; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008565:Membrane Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D014747:Vinblastine; D016572:Cyclosporine; D014700:Verapamil; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D002211:Capsaicin