ATP-dependent transport of glutathione S-conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2

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Abstract

The membrane proteins mediating the ATP-dependent transport of glutathione S-conjugates and related amphiphilic anions have been identified as the multidrug resistance proteins MRP1 and MRP2. These 190-kDa membrane glycoproteins were cloned in recent years and shown to be unidirectional, ATP-driven, export pumps with an amino acid identity of 49% in humans. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes; whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, such as the hepatocyte canalicular membrane and kidney proximal tubule luminal membrane. Physiologically important substrates of both transporters include glutathione S-conjugates, such as leukotriene C4, as well as bilirubin glucuronides, 17β-glucuronosyl estradiol and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 studied in inside-out oriented membrane vesicles is very different from MDR1 P-glycoprotein. MRP1 and MRP2 may be termed conjugate transporting ATPases, functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.

Introduction

Glutathione S-conjugate formation of endogenous lipophilic compounds and xenobiotics often precedes their transport across cellular membranes 1, 2. The transport of the conjugates into the extracellular space has been characterized as a primary-active, ATP-dependent transport by studies using inside-out oriented plasma membrane vesicles from many cell types including erythrocytes 3, 4and hepatocytes 5, 6, 7, 8, 9. Identification of the membrane proteins mediating the ATP-dependent unidirectional transport of glutathione S-conjugates was initiated by the characterization of the 190-kDa membrane glycoprotein mediating the ATP-dependent export of the endogenous glutathione S-conjugate leukotriene C4 (LTC4) [10]. These studies resulted in the elucidation of the function of the multidrug resistance protein (MRP1) as a primary-active ATP-dependent membrane transporter for leukotriene C4 and many other amphiphilic anionic conjugates 11, 12, 13, 14, 15, 16.

The multidrug resistance protein, MRP or MRP1 [17], is a membrane glycoprotein of an apparent molecular mass of 190 kDa that was originally identified on the basis of its overexpression in multidrug-resistant tumor cell lines 17, 18. The original sequence analysis of MRP1 indicated that it is a member of the ATP-binding cassette (ABC) superfamily of transporters [17]. An isoform of MRP1 with a similar substrate specificity and a distinct, although related, sequence has been cloned more recently and localized predominantly to the hepatocyte canalicular membrane 19, 20, 21, 22, 23but also to the apical membrane of kidney proximal tubule epithelia [24]. This isoform has been termed MRP2, or canalicular MRP (cMRP), or canalicular multispecific organic anion transporter (cMOAT). MRP2 mediates the ATP-dependent transport of glutathione S-conjugates and glucuronides across apical membrane domains 23, 25. Both MRP1 and MRP2 may be termed conjugate transporting ATPases with a broad substrate specificity. Additional members of the MRP family of ABC transporters have been identified recently [26].

Section snippets

Comparison of MRP1 and MRP2

Human MRP1 and MRP2 are composed of 1531 and 1545 amino acids, respectively, both contain two characteristic ATP-binding domains and both are members of the MRP family of transporters within the superfamily of ABC transporters 17, 23, 27(Table 1). Human MRP1 and MRP2 exhibit an amino acid identity of 49%, with the highest degree of amino acid identity in the carboxyl-terminal domain and in both nucleotide-binding domains 19, 23. The molecular mass of unglycosylated MRP1 and MRP2 is 172 [17]and

Substrate specificity of the conjugate export pumps MRP1 and MRP2

The substrate specificity of recombinant human MRP1 determined in membrane vesicles from transfected cells is indistinguishable from that determined in inside-out membrane vesicles from drug-selected MRP1-overexpressing cells, such as the HL60/ADR line 11, 12, 14. The endogenous glutathione S-conjugate and mediator LTC4 is the substrate with the highest affinity known at present, whereas ATP-dependent transport of oxidized glutathione (GSSG) proceeds with an apparently low affinity, with a Km

Role of MRP1 and MRP2 in detoxification and defense against oxidative stress

Biosynthesis and export from cells of conjugates of endogenous and xenobiotic lipophilic substances with glutathione, glucuronate or sulfate is of vital importance in detoxification and cellular homeostasis. The broad substrate specificity of the conjugate export pumps MRP1 and MRP2 enables the terminal excretion of a multitude of conjugates and amphiphilic anions which are formed by a large number of relatively specific monooxygenases and transferases in phase I and phase II metabolism of

Mutants Lacking MRP2 or MRP1

The human Dubin–Johnson syndrome represents an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into bile [45]. Identification of the human canalicular membrane protein mediating the ATP-dependent transport of glutathione and glucuronate conjugates as MRP2 suggested that the absence of this transporter is the molecular basis of this hereditary disease 19, 46. Selective detection of human MRP2 by an antibody directed against the carboxyl-terminal sequence of

Acknowledgements

Work in the authors’ laboratory was supported in part by the Deutsche Forschungsgemeinschaft, Bonn and by the Forschungsschwerpunkt Transplantation Heidelberg.

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