title: Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).
abstract: The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.|Gene Medicine Research Group, Nuffield Department of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford , Oxford OX3 9DU, U.K.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Pluchino
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Hall
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Janna K
authors.initials: JK
authors.lastname: Moen
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Eduardo E
authors.initials: EE
authors.lastname: Chufan
authors.affiliation: Laboratory of Pathology, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Fetsch
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Suneet
authors.initials: S
authors.lastname: Shukla
authors.affiliation: Gene Medicine Research Group, Nuffield Department of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford , Oxford OX3 9DU, U.K.
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Gill
authors.affiliation: Gene Medicine Research Group, Nuffield Department of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford , Oxford OX3 9DU, U.K.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Hyde
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Di
authors.initials: D
authors.lastname: Xia
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002461:Cell Line, Transformed; D019008:Drug Resistance, Neoplasm; D005456:Fluorescent Dyes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007915:Lepidoptera; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D049990:Membrane Transport Modulators; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C095122:multidrug resistance protein 3
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