Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, 1113 Budapest, Hungary.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2008; 283(38):26059-70. DOI: 10.1074/jbc.M803230200
Source: PubMed


Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

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Available from: Csilla Hegedüs, Feb 14, 2014
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    • "In order to compare the relative expression of ABCG2 in the red cell membrane to those in known expression systems, we performed Western blot experiments using isolated red cell membranes, isolated Sf9 insect cell membranes expressing the human ABCG2 protein, and A431 cells overexpressing ABCG2 (see Supplementary Materials and refs. [45], [46], [47]. In accordance with previous data in the literature [35], [36], [37], [38], [39], we detected both the monomeric and dimeric forms of ABCG2 in the red cell membrane but found that this assay is not suitable for the proper quantitation of small changes in ABCG2 expression (see Supplementary Materials). "
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    ABSTRACT: We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.
    PLoS ONE 11/2012; 7(11):e48423. DOI:10.1371/journal.pone.0048423 · 3.23 Impact Factor
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    • "The 5D3 antibody recognizes a conformationsensitive extracellular epitope in BCRP [32]. Binding of the 5D3 antibody to the surface of cells expressing wild-type BCRP or C603A was examined using flow cytometry as previously described [33]. "
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    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 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, 5D3 antibody binding, and 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 a dimer/oligomer in vivo in intact cells using the FRET technique. We have also shown that Cys(603) alone does not seem to be essential for dimer/oligomer formation of BCRP.
    International Journal of Biochemistry and Molecular Biology 01/2010; 1(1):1-11.
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    ABSTRACT: The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and organic anions across the plasma membrane. Hydropathy analysis suggests that BCRP consists of a nucleotide-binding domain (residues approximately 1-395) and a membrane-spanning domain (MSD) (residues approximately 396-655); however, its exact topology structure remains unknown. In this study, we determined the topology structure of BCRP by inserting hemagglutinin (HA) tags in its predicted hydrophilic regions of the MSD. HA-tagged BCRP mutants were expressed in HEK cells and tested for their ability to efflux mitoxantrone and BODIPY-prazosin. Polarity of the inserted tags with respect to the plasma membrane was determined by immunofluorescence. All of the mutants were expressed at levels comparable to wild-type BCRP as revealed by immunoblotting with specific antibodies against BCRP and the HA tag. Insertions at residues 423, 454, 462, 499, 529, 532, and 651 produced functional mutants, whereas insertions at residues 560, 594, and 623 resulted in mutants with significantly reduced activity and insertions at residues 387, 420, 474, and 502 completely abrogated the activity. HA tags inserted at residues 387, 474, 529, 532, 560, and 651 were localized intracellularly, whereas those inserted at residues 420, 423, 454, 499, 502, 594, and 623 revealed an extracellular location. Residue 462 was localized in a transmembrane (TM) segment. These results provide the first direct experimental evidence in support of a 6-TM model for BCRP with the amino and carboxyl termini of the MSD located intracellularly. These data may have important implications for understanding the transport mechanism of BCRP.
    Biochemistry 01/2009; 47(52):13778-87. DOI:10.1021/bi801644v · 3.02 Impact Factor
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