Identification of the Multidrug‐Resistance Protein (MRP) as the Glutathione‐S‐Conjugate Export Pump of Erythrocytes

Abstract

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C, (LTC,), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the K,,, value for LTC, of 11 8 2 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC, revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.Keywords: ATP-dependent transport; erythrocytes ; glutathione conjugate ; multidrug-resistance protein ; photoaffinity labeling.The export of glutathione S-conjugates from erythrocytes has been functionally known for many years [1-61. Using insideout oriented vesicles of erythrocyte membranes, transport of glutathione S-conjugates and oxidized glutathione has been shown to be a primary-active ATP-dependent process [2, 6-91. Similar transport processes have been found in other tissues [lo-171, though erythrocytes are an especially convenient model system for studying this transport since they lack y-glutamyltransferase and they do not have intracellular membranes. Therefore, further metabolism of glutathione S-conjugates is avoided and preparation of pure inside-out plasma membrane vesicles is facilitatedThe most widely used glutathione S-conjugate is S-(2,4-dinitropheny1)glutathione (Dnp-SG). In several tissues, the endogenous mediator leukotriene C, (LTC,) was identified as the substrate with the highest affinity for this transport system [13, 16, 171, but until now its transport across the erythrocyte membrane has not been described. Attempts to identify the protein responsible for this transport process in erythrocytes yielded several candidate proteins with apparent molecular masses of 23-90 kDa [18-201. However, in none of these studies was a direct involvement of the respective protein in glutathione S-conjugate transport proven. PI.Correspondence to D. Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrm, Im Neuenheimer Feld 280, D-69120 Heidelberg, GermanyAbbreviations. MRP, multidrug resistance protein (multidrug resistance-associated protein); LTC,, leukotriene C,: Dnp-SG, S-(2,4-dinitro- Recently, MRP was found to function as an AT...

Regulation of transbilayer plasma membrane phospholipid asymmetry

“…Like ABCA1 and ABCB1, ABCB4 may be involved in lipid efflux rather than in the maintenance of plasma membrane lipid asymmetry. ABCC1 (MRP1) was identified initially as a conjugated glutathione transporter (131,132). This enzyme was also shown to catalyze the efflux of short-chain NBD phospholipid analogs, but not endogenous lipids (such as PS), from the inner to the outer monolayer of the erythrocyte (57,133,134) and may account for the previously described efflux activity of these lipids (67,112).…”

Interaction of Positional Isomers of Quercetin Glucuronides with the Transporter ABCC2 (cMOAT, MRP2)

“…MK571 has been used extensively as an inhibitor of ABCC2, e.g. (Pulaski et al, 1996), and cyclosporin is also an inhibitor (Wortelboer et al, 2003). We used a combination of quercetin conjugates and inhibitors to test interaction with ABCC2 and to assess the predictive power of an in silico generated 3D model of ABCC2.…”

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

“…Human erythrocytes were previously demonstrated to constitute a good model to study the transport activity of MRP1 [33,39,[44][45][46]. In this study, we showed that the flavones apigenin and acacetin and the flavonols morin and myricetin were effective inhibitors of MRP1-mediated BCECF efflux from human erythrocytes and also from the MRP1-expressing resistant cancer cell line MDA-MB-231.…”

“…The expression of MRP1 [39], MRP4 [40] and MRP5 [41] has thusfar been demonstrated for this cell type. Western blot analysis of erythrocyte membrane preparations performed by Rychlik et al [33] revealed a much higher expression of MRP1 than MRP5, and no P-glycoprotein was recorded in erythrocytes.…”