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J. Biol. Chem., Vol. 281, Issue 20, 13906-13914, May 19, 2006

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Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)^*

Alice Rothnie, Richard Callaghan, Roger G. Deeley, and Susan P. C. Cole¹

From the Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada and the Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (K_d) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

Received for publication, January 27, 2006 , and in revised form, March 15, 2006.

^* This work was supported by the Canadian Institutes of Health Research (Grant MOP-10519). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Canada Research Chair in Cancer Biology. To whom correspondence should be addressed: Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2636; Fax: 613-533-6830; E-mail: SusanPC.Cole{at}Queensu.ca.

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This article has been cited by other articles:

				K. Maeno, A. Nakajima, G. Conseil, A. Rothnie, R. G. Deeley, and S. P. C. Cole Molecular Basis for Reduced Estrone Sulfate Transport and Altered Modulator Sensitivity of Transmembrane Helix (TM) 6 and TM17 Mutants of Multidrug Resistance Protein 1 (ABCC1) Drug Metab. Dispos., July 1, 2009; 37(7): 1411 - 1420. [Abstract] [Full Text] [PDF]

				A. Rothnie, G. Conseil, A. Y. T. Lau, R. G. Deeley, and S. P. C. Cole Mechanistic Differences between GSH Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) and GSH Modulation of MRP1-Mediated Transport Mol. Pharmacol., December 1, 2008; 74(6): 1630 - 1640. [Abstract] [Full Text] [PDF]