Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family

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Introduction

Membrane proteins mediating the ATP-dependent transport of lipophilic compounds conjugated to glutathione, glucuronate, or sulfate have been recognized as members of the multidrug resistance protein (MRP) family1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. In this article we focus on three human MRP family members, MRP1, MRP2, and MRP3, encoded by genes on different chromosomes7, 8, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. The amino acid identity between these isoforms is 48% between MRP1 and MRP2, 58% between MRP1 and MRP3, and 46% between MRP2 and MRP316, 20, 21. MRP1 has been localized to the plasma membrane of a variety of non-polarized cells, whereas MRP2 and MRP3 have distinct domain-specific localizations in polarized cells7, 8, 21, 22. In hepatocytes21, 24and proximal tubule cells of the kidney[25]MRP2 is localized exclusively to the apical membrane domain, and has, therefore, been termed apical conjugate export pump[20]. The isoform MRP3 has been localized to the basolateral membrane domain of hepatocytes21, 22. MRP3 may function as a compensatory conjugate export pump under conditions of MRP2 deficiency, such as human Dubin–Johnson syndrome which is due to the absence of the MRP2 protein from the hepatocyte canalicular membrane because of mutations in the MRP2 gene23, 24, 26, 27, 28, 29.

Expression of recombinant MRP2 at high levels has been achieved in HEK293 cells[8]. In these transfected cells, the MRP2 protein was localized only in part to the plasma membrane. Human MRP2 overexpressed in HEK293 cells enhanced the resistance to several cytotoxic anticancer drugs. These results provide the first direct evidence that MRP2 confers resistance to cytotoxic drugs[8].

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Substrates for transport assays

3H-labeled substrates, including LTC4 and 17β-glucuronosyl estradiol, were from Du Pont–New England Nuclear (Boston, MA). [3H]Monoglucuronosyl bilirubin was synthesized enzymatically using microsomes from UDPglucuronosyl transferase 1A1-transfected HeLa cells[30]and UDP-[3H]glucuronate from Biotrend (Köln, Germany) in a procedure described recently30, 31. [3H]MGB was stabilized with ascorbate and stored under argon[31]. The fluorescent penta-anion Fluo-3 was measured by its fluorescence in the

Comparison of the genes encoding the human multidrug resistance proteins MRP1, MRP2, and MRP3

The family of human multidrug resistance proteins comprises at least six members known as MRP1 (symbol ABCC1)14, 36, MRP2 (ABCC2; also known as canalicular MRP15, 26, 27, 37or cMOAT16, 23), MRP3 (ABCC3)9, 17, 18, 19, 21, 22, and the currently less well characterized isoforms MRP4 through MRP617, 18. MRP1, MRP2, and MRP3 genes have been localized to chromosomes 16p13[14], 10q24[16], and 17q2117, 18, respectively. Elucidation of the complete exon–intron organization of the genes encoding MRP1[34]

Summary

The ATP-dependent transport of lipophilic substances conjugated with glutathione, glucuronate, or sulfate is mediated by members of the multidrug resistance protein (MRP) family. MRP1, MRP2, and MRP3 (symbols ABCC1, ABCC2, and ABCC3, respectively) have overlapping and similar substrate specificities but differ markedly in their kinetic properties. Prototypic high-affinity substrates include the glutathione S-conjugate leukotriene C4, 17β-glucuronosyl estradiol, and glucuronosyl bilirubin. All

Acknowledgements

Parts of this work were supported by grants from the Deutsche Forschungsgemeinschaft (SFB 352/B3 and SFB 601/A2), by the Tumorzentrum Heidelberg/ Mannheim, by the Fonds der Chemischen Industrie, Frankfurt, and by the Alexander-von-Humboldt-Stiftung, Bonn.

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