Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

Paradigms and Technologies
Methods Development

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

Pluchino, Kristen M; Hall, Matthew; Moen, Janna K; Chufan, Eduardo E; Fetsch, Patricia A; Shukla, Suneet; Gill, Deborah R; Hyde, Stephen C; Xia, Di; Ambudkar, Suresh V; Gottesman, Michael M;

Keywords

  • Animals
  • Antineoplastic Agents/ pharmacology
  • Biological Transport/ drug effects
  • Cell Line
  • Cell Line, Transformed
  • Drug Resistance, Neoplasm
  • Fluorescent Dyes/ metabolism
  • HeLa Cells
  • Humans
  • Kinetics
  • Lepidoptera
  • Membrane Transport Modulators/ pharmacology
  • Mice
  • Models, Molecular
  • P-Glycoproteins/ antagonists & inhibitors
  • P-Glycoproteins/ chemistry
  • P-Glycoproteins/ genetics
  • P-Glycoproteins/ metabolism
  • Peptide Fragments/ antagonists & inhibitors
  • Peptide Fragments/ chemistry
  • Peptide Fragments/ genetics
  • Peptide Fragments/ metabolism
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins/ chemistry
  • Recombinant Fusion Proteins/ genetics
  • Recombinant Fusion Proteins/ metabolism
  • Recombinant Proteins/ chemistry
  • Recombinant Proteins/ genetics
  • Recombinant Proteins/ metabolism

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