Abstract
The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.
Highlights
The ABCG21 (MXR/BCRP/ABCP) protein causes multidrug resistance in cancer cells and may have an important function
The substrate specificity of ABCG2 partially overlaps with the other major multidrug resistance ABC transporters, MDR1 and human multidrug resistance protein 1 (MRP1); the compounds transported by ABCG2 are large, hydrophobic molecules, including mitoxantrone, topotecan, flavopiridol, methotrexate, and Hoechst 33342 [13, 16, 17]
In the present experiments we have studied the interaction of the anti-ABCG2 monoclonal antibody 5D3 in various cell types expressing the human ABCG2 protein and examined the effects of ABCG2 protein modulators on this interaction
Summary
The ABCG21 (MXR/BCRP/ABCP) protein causes multidrug resistance in cancer cells and may have an important function. When the effects of different agents were investigated the membranes were preincubated in assay mix containing 2 mM sodium orthovanadate, 1 M Ko143, 10 mM MgAMP, MgADP, MgAMP-PNP, MgATP, or 10 mM AMP, ADP, AMP-PNP, ATP plus 2 mM EDTA, or the combination of these agents (as described, panels C and D) for 5 min at 37 °C before the addition of the 5D3 antibody.
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