Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter.
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.
Orbán
Tamás I
TI
Research Group for Membrane Biology, Hungarian Academy of Sciences, Diószegi u. 64, 1113 Budapest, Hungary.
Seres
László
L
Ozvegy-Laczka
Csilla
C
Elkind
N Barry
NB
Sarkadi
Balázs
B
Homolya
László
L
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000493:pharmacokinetics; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001562:Benzimidazoles; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; C070990:vanadate-sensitive ATPase; C017807:bisbenzimide ethoxide trihydrochloride