Mechanistic Differences between GSH Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) and GSH Modulation of MRP1-Mediated Transport □ S Alice Rothnie, 1 Gwenae ¨ lle Conseil, Andrea Y. T. Lau, Roger G. Deeley, and Susan P. C. Cole Division of Cancer Biology & Genetics, Queen’s University Cancer Research Institute, Kingston, Ontario, Canada, Received May 21, 2008; accepted September 2, 2008 ABSTRACT Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP- dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mecha- nism is multifaceted, especially with respect to the partici- pation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH- associated substrates/modulators on the binding and hy- drolysis of ATP by MRP1 using 8-azidoadenosine-5'-[ 32 P]- triphosphate ([ 32 P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing deriva- tive S-methylGSH (S-mGSH), but none of the GSH-associ- ated substrate/modulators, caused a significant increase in [- 32 P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trap- ping of [- 32 P]azidoADP. [- 32 P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verap- amil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/ modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences be- tween MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport. The increased expression of multidrug resistance protein 1 (MRP1/ABCC1) in tumor cells causes resistance to chemo- therapy (Cole et al., 1992; Deeley et al., 2006). MRP1, a member of the ATP-binding cassette (ABC) superfamily of transporters, which is also expressed in almost all normal tissues, uses the energy from ATP binding and hydrolysis to efflux a wide variety of drugs (e.g., anthracyclines, plant alkaloids, and antifolates) across the plasma membrane. In addition to transporting drugs, MRP1 actively effluxes many endogenous conjugated organic anions and metabolites of xenobiotics, and thus plays a physiological and a protective role in both normal and malignant tissues (Wijnholds et al., 2000; Leslie et al., 2005). One striking feature of the transport mechanism of MRP1 function is its complex interaction(s) with the reducing trip- eptide GSH. MRP1 mediates the cellular efflux of many GSH conjugates (Fig. 1A), including the proinflammatory cystei- nyl leukotriene C 4 (LTC 4 ), which has been established to be a major physiological substrate of MRP1 (Loe et al., 1996b; Wijnholds et al., 1997). It also transports the GSH-conju- gated metabolites of many xenobiotics and the pro-oxidant glutathione disulfide (GSSG) (Leier et al., 1996; Haimeur This work was supported by grant MOP-10519 from the Canadian Insti- tutes of Health Research (CIHR). A.R. was supported by a CIHR postdoctoral fellowship. S.P.C.C. is the Canada Research Chair in Cancer Biology and Bracken Chair in Genetics and Molecular Medicine. 1 Current affiliation: University of Warwick, Department of Biological Sci- ences, Gibbet Hill Campus, Coventry, United Kingdom. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.049080. □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: MRP1, multidrug resistance protein 1; ABC, ATP binding cassette; S-mGSH, S-methyl glutathione; GSSG, glutathione disulfide; LTC 4 , leukotriene C 4 ;E 2 17G, estradiol glucuronide; MSD, membrane-spanning domain; NBD, nucleotide binding domain; DTT, dithiothreitol; azidoATP, 8-azidoadenosine triphosphate; Vi, orthovanadate; PAGE, polyacrylamide gel electrophoresis; NBS, nucleotide binding site; [- 32 P]azidoATP, 8-azidoadenosine-5'-[- 32 P]triphosphate; [- 32 P]azidoATP, 8-azidoadenosine-5'-[- 32 P]triphosphate. 0026-895X/08/7406-1630 –1640$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 6 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 49080/3404400 Mol Pharmacol 74:1630–1640, 2008 Printed in U.S.A. 1630 http://molpharm.aspetjournals.org/content/suppl/2008/09/02/mol.108.049080.DC1 Supplemental material to this article can be found at: at ASPET Journals on July 18, 2018 molpharm.aspetjournals.org Downloaded from