Purification of breast cancer resistance protein ABCG2 and role of arginine-482 |
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Authors: | A Pozza J M Perez-Victoria A Sardo A Ahmed-Belkacem A Di Pietro |
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Institution: | (1) Institut de Biologie et Chimie des Protéines, UMR5086 CNRS-Université de Lyon et IFR128 BioSciences Lyon-Gerland, 7 Passage du Vercors, 69367 Lyon Cedex 07, France;(2) Instituto de Parasitologia y Biomedicina ‘Lopez-Neyra’, Consejo Superior de Investigaciones Cientificas, Granada, Spain |
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Abstract: | Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate,
and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding
capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered
sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated
a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding
of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound
similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic
inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound
similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug
transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency
is not mediated by changes in drug binding.
Received 10 April 2006; received after revision 22 May 2006; accepted 12 June 2006
A. Pozza and J. M. Perez-Victoria contributed equally to this work |
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Keywords: | Breast cancer resistance protein multidrug resistance ABC transporter substrates inhibitors ATPase activity anticancer chemotherapy |
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