Int J Biochem Mol Biol 2010;1(1):1-11
Original Article
Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer
formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells
Zhanglin Ni, Michelle E. Mark, Xiaokun Cai, Qingcheng Mao
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington 98195-7610, USA.
Received January 19, 2010; accepted January 28, 2010; available online February 12, 2010; published August 1, 2010
Abstract: The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an
important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been
demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type
BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein by mutagenesis and expressed in HEK293 cells by
transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by
immunoblotting, the 5D3 antibody binding, flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was
analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at
comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the
same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data
consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form dimer/oligomer in vivo in intact
cells using the FRET technique. We have also shown that Cys603 alone does not seem to be essential for dimer/oligomer formation of
BCRP.(IJBMB1001001).
Keywords: BCRP, ABCG2, dimerization, oligomerization, and FRET
Full Text PDF
Address all correspondence to:
Qingcheng Mao, PhD
Department of Pharmaceutics
School of Pharmacy
Box 357610, University of Washington
Seattle, WA 98195-7610
Tel: (206) 685-0355; Fax: (206) 543-3204
Email: qmao@u.washington.edu
Original Article
Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer
formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells
Zhanglin Ni, Michelle E. Mark, Xiaokun Cai, Qingcheng Mao
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington 98195-7610, USA.
Received January 19, 2010; accepted January 28, 2010; available online February 12, 2010; published August 1, 2010
Abstract: The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an
important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been
demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type
BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein by mutagenesis and expressed in HEK293 cells by
transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by
immunoblotting, the 5D3 antibody binding, flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was
analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at
comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the
same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data
consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form dimer/oligomer in vivo in intact
cells using the FRET technique. We have also shown that Cys603 alone does not seem to be essential for dimer/oligomer formation of
BCRP.(IJBMB1001001).
Keywords: BCRP, ABCG2, dimerization, oligomerization, and FRET
Full Text PDF
Address all correspondence to:
Qingcheng Mao, PhD
Department of Pharmaceutics
School of Pharmacy
Box 357610, University of Washington
Seattle, WA 98195-7610
Tel: (206) 685-0355; Fax: (206) 543-3204
Email: qmao@u.washington.edu
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