Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter

Background & Purpose: Drug‐resistant cancer cells frequently display efflux pumps such as P‐glycoprotein (P‐gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. These transporters are each capable of mediating the active efflux of numerous anticancer drugs and display relatively distinct substrate preferences. The last, most recently discovered member, ABCG2, plays a major role in resistance in several types of cancer and the precise pharmacology of this multidrug transporter remain unresolved as does the nature of substrate binding. Experimental Approach: Plasma membranes from insect cells expressing ABCG2 were used to characterise binding of [ 3 H]daunomycin to the multidrug transporter. The kinetics of association and dissociation for this substrate and several other compounds were also determined in this experimental system. Key Results: The dissociation constant for [ 3 H]daunomycin binding was 564±57 nM and a Hill slope of 1.4 suggested cooperative binding. Doxorubicin, prazosin and daunomycin completely displaced the binding of radioligand, while mitoxantrone and Hoechst 33342 produced only a partial displacement. Analysis of the dissociation rates revealed that [ 3 H]daunomycin and doxorubicin bind to multiple sites on the transporter. Conclusions: Both kinetic and equilibrium data support the presence of at least two symmetric drug binding sites on ABCG2, which is distinct from the asymmetry observed for P‐gp. The data provide the first molecular details underlying the mechanism by which this transporter is capable of interacting with multiple substrates. British Journal of Pharmacology (2006) 149 , 506–515. doi: 10.1038/sj.bjp.0706904