Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

Tamás I Orbán; László Seres; Csilla Ozvegy-Laczka; N Barry Elkind; Balázs Sarkadi; László Homolya

doi:10.1016/j.bbrc.2007.12.172

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

Biochem Biophys Res Commun. 2008 Mar 14;367(3):667-73. doi: 10.1016/j.bbrc.2007.12.172. Epub 2008 Jan 7.

Authors

Tamás I Orbán¹, László Seres, Csilla Ozvegy-Laczka, N Barry Elkind, Balázs Sarkadi, László Homolya

Affiliation

¹ Research Group for Membrane Biology, Hungarian Academy of Sciences, Diószegi u. 64, 1113 Budapest, Hungary.

PMID: 18182157
DOI: 10.1016/j.bbrc.2007.12.172

Abstract

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

ATP Binding Cassette Transporter, Subfamily G, Member 2
ATP-Binding Cassette Transporters / chemistry
ATP-Binding Cassette Transporters / genetics
ATP-Binding Cassette Transporters / metabolism*
Adenosine Triphosphatases / chemistry
Adenosine Triphosphatases / drug effects
Adenosine Triphosphatases / metabolism
Animals
Benzimidazoles / pharmacokinetics
Cell Line
Cell Membrane / chemistry
Cell Membrane / metabolism
Chlorocebus aethiops
Flow Cytometry
Gene Transfer Techniques
Green Fluorescent Proteins / genetics
Humans
Kidney / cytology
Kidney / metabolism
Neoplasm Proteins / chemistry
Neoplasm Proteins / genetics
Neoplasm Proteins / metabolism*
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Spodoptera

Substances

ABCG2 protein, human
ATP Binding Cassette Transporter, Subfamily G, Member 2
ATP-Binding Cassette Transporters
Benzimidazoles
Neoplasm Proteins
Recombinant Fusion Proteins
Green Fluorescent Proteins
Adenosine Triphosphatases
vanadate-sensitive ATPase
bisbenzimide ethoxide trihydrochloride