Review
Transport of glutathione and glutathione conjugates by MRP1

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Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene C4) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.

Introduction

The metabolism, distribution and elimination of xenobiotics and their metabolites are accomplished by a complex array of pathways involving multiple biotransformation and conjugation enzymes, endogenous nucleophiles and membrane transport proteins. Phase I metabolism refers to the biotransformation of xenobiotics to electrophilic intermediates and is mediated primarily by the cytochrome P450 enzymes and other mixed-function oxidases. These intermediates might then be further modified by conjugation to small endogenous nucleophiles such as glutathione (GSH), glucuronate and sulfate by reactions that are collectively known as phase II metabolism. In some cases, xenobiotics are sufficiently electrophilic to undergo conjugation without prior modification by phase I enzymes. In either case, phase II conjugation reactions are almost always catalysed by a transferase enzyme. Conjugation to GSH can occur spontaneously in some instances but it is typically catalysed by one or more members of several different families of GSH S-transferases (GSTs) [1]. Conjugation reactions usually render electrophilic xenobiotic intermediates less reactive and, in most cases, reduce or eliminate their pharmacological and/or toxic actions. There are, however, some important exceptions [2]. Regardless of whether products of phase II metabolism remain biologically active, however, these conjugated organic anions must be exported from cells so that they can be eliminated from the body.

In addition to their role in xenobiotic metabolism, GSH conjugation reactions are important in normal physiological processes. Thus, several endogenous arachidonic acid derivatives exist as GSH conjugates, one of which – the cysteinyl leukotriene (LT)C4 – is an important mediator of inflammation. LTC4 is formed through the conjugation of GSH to LTA4 by LTC4 synthase [IUBMB (http://www.chem.qmul.ac.uk/iubmb/ ) enzyme nomenclature EC 4.4.1.20]. To exert its pro-inflammatory actions, however, LTC4 must be exported from the cells in which it is synthesized so that it can be converted into its active metabolites LTD4 and LTE4, which then bind to their respective receptors in the plasma membrane of cells in their target tissues. For many years, it was unclear how LTC4 and other anionic conjugates of xenobiotics and endogenous metabolites crossed the plasma membrane to the extracellular milieu, although there was strong but indirect evidence that an energy-dependent process was involved. It was postulated that GSH conjugates were exported from cells by a GS-X pump, by a process dubbed the phase III elimination step of drug metabolism [3]. At the time, however, the number of such pumps and their molecular characteristics were not known.

Over the past decade, it has been established that the active cellular efflux of GSH conjugates and other conjugated metabolites is mediated primarily by a subset of proteins belonging to the ATP-binding cassette (ABC) superfamily of transport proteins (http://nutrigene.4t.com/humanabc.htm ). In humans, the ABC gene superfamily contains 49 members, which are organized into seven subfamilies (A–G) based on their relative sequence homology to one another [4]. Several members of the ABC subfamily C [ABCC, also known as the multidrug resistance protein (MRP)/CFTR (cystic fibrosis transmembrane conductance regulator) subfamily] mediate the cellular efflux of GSH conjugates and GSH itself, at least in vitro 5, 6, 7, 8. This review focuses mainly on MRP1 (gene symbol ABCC1) because knowledge of the molecular mechanisms and physiological functions of this transporter that are related to GSH and GSH conjugate transport is the most advanced for all of the MRP-related proteins. Thus, the experimental approaches for enhancing the understanding of the molecular basis of MRP1 function will be useful in the study of other ABCC transporters.

Section snippets

The MRP-related GSH and GSH conjugate transporters

As with all ABC proteins, the MRP-related transporters have a common core structure that contains two membrane-spanning domains (MSDs), each comprising six transmembrane (TM) α-helices and two nucleotide-binding domains (NBDs) (Figure 1). Each highly conserved NBD contains three conserved sequence motifs: the Walker A (WA) and Walker B (WB) motifs, which are found in many ATPases, and the ABC family signature sequence LSGGQ (or C motif), which is found only in ABC proteins. The activity of ABC

MRP1

MRP1 (ABCC1) was the first of the xenobiotic-transporting MRP-related proteins to be cloned and was identified based on its overexpression in a multidrug-resistant lung cancer cell line [14]. The 190-kDa MRP1 is highly overexpressed in many drug-resistant tumour cell lines and, thus far, it is the only MRP-related transporter protein for which a substantial body of evidence indicates that it contributes to the failure of chemotherapy in some malignant diseases [12]. MRP1 is expressed at

Concluding remarks

This review has been limited to the consideration of the role of MRP1 in GSH and GS-X transport, but it is well established that the MRP1 homologue MRP2 (gene symbol ABCC2) can also transport a comparable spectrum of organic anions 78, 79, 80, 81, 82, 83. The important difference is that MRP2-mediated transport occurs across the apical membranes of cells and in a more limited number of tissues (i.e. liver, kidney, placenta and intestine). Thus, MRP2 has physiological and pharmacological

Update

The work referred to in the text as (A.J. Rothnie et al., unpublished) is now published [93].

Acknowledgements

We thank Maureen Hobbs and Alice Rothnie for their assistance in the preparation of the manuscript and figures, and the Canadian Institutes of Health Research for grant support. S.P.C.C. holds the Canada Research Chair in Cancer Biology, and R.G.D. is the Queen's University Stauffer Professor of Basic Cancer Research.

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