Abstract
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation 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 hydrolysis of ATP by MRP1 using 8-azidoadenosine-5′-[32P]-triphosphate ([32P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [γ-32P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [α-32P]azidoADP. [α-32P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, 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 between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.
Footnotes
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This work was supported by grant MOP-10519 from the Canadian Institutes 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.
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ABBREVIATIONS: MRP1, multidrug resistance protein 1; ABC, ATP binding cassette; S-mGSH, S-methyl glutathione; GSSG, glutathione disulfide; LTC4, leukotriene C4; E217βG, 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; [γ-32P]azidoATP, 8-azidoadenosine-5′-[γ-32P]triphosphate; [α-32P]azidoATP, 8-azidoadenosine-5′-[α-32P]triphosphate.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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↵1 Current affiliation: University of Warwick, Department of Biological Sciences, Gibbet Hill Campus, Coventry, United Kingdom.
- Received May 21, 2008.
- Accepted September 2, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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