The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD 0 ) connected to the core by a cytoplasmic linker (L 0 ). We have studied truncated MRP1 versions containing either the MDRlike core alone or the core plus linker L 0 , produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Fulllength MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C 4 and N-ethylmaleimide glutathione. In addition, leukotriene C 4 -stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L 0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD 0 region but still containing L 0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD 0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD 0 L 0 and the MRP1 minus TMD 0 L 0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD 0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane. MDR1 P-glycoprotein (MDR1 Pgp)1 and MRP1 (multidrug resistance protein 1) are members of the ATP binding cassette (ABC) transporter family that can cause multiple drug resistance in tumor cells. MDR1 Pgp is an ATP-dependent drug extrusion pump and confers resistance to a wide variety of hydrophobic toxic agents (1). MRP1 has been shown to be a high affinity primary active transporter for the glutathioneconjugated eicosanoid, leukotriene C 4 (LTC 4 ) (2, 3) and to transport various other compounds that are conjugated to glutathione, sulfate, or glucuronide (2, 4 -6). The physiological functions of MRP1 range from the mediation of an inflammatory response to the elimination of certain xenobiotics (7-11), and this protein may play a role in the chemotherapy resistance of several types of cancer (12).Vanadate inhibits ATP-dependent drug transport both by MDR1 and MRP1 (1, 7), and in the presence of vanadate, the trapping of an adenine nucleotide in these proteins has been demonstrated (13,14). Transported compounds specifically increase the rate of vanadate-dependent nucleotide occlusion in MRP1 (14), similarly to what has been shown for hydrophobic drugs in the case of MDR1 Pgp (15). Vanadate-dependent, drug-stimulated nucleotide trapping reflects a partial reaction of the multidrug transporters and thus can be used to examine their functional characteristics.MDR1 Pgp and MRP1 share a similar core structure, consisting of a tandem repeat of transmembrane domains (TMDs) and cytoplasmic ABC-containing regions. However,...

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions. We expressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles. Both MRP1 and MRP2 actively transported leukotriene C 4 and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1. Methotrexate was actively transported by both proteins, although more efficiently by MRP2. ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions. Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimulated, MRP2-mediated NEM-GS uptake. Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concentrations in MRP2. ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate. Probenecid, sulfinpyrazone, indomethacin, furosemide, and penicillin G all significantly increased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1. These results indicate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2. Our data suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein.Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membranebound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRPlike ATP-binding cassette transporters.Overexpression of the multidrug transporter proteins, Pglycoprotein (MDR1) 1 or the multidrug resistance-associated protein (MRP) provides the molecular basis of the multidrug resistance phenotype in tumor cells. The possible clinical importance fuels an intensive research activity toward a better understanding of the molecular structure and mechanism of action of these membrane transporters (1-3).Both P-glycoprotein and MRP, together with several other bacterial and eukaryotic transporters, are members of the ABC-transporter (ATP-binding cassette) protein family. These proteins share a common molecular architecture, i.e. they contain two large transmembrane domains and two cytoplasmic ATP utilization (ABC) units (4). Due to the difficulty of crystallizing large membrane proteins, no detailed three-dimensional structure of any members of these transporters is currently available, and empirical prediction methods are used to obtain molecular models of their structure, especially to predict the locations and numbers of the membrane-spanning helices. In most cases, these methods identify six short transmembrane segments in each of the two transmembrane domains (1-4). The relevance of the prediction for the membrane topology of CFTR has been confirmed experimentally by insertional mutagenesis (5), thus proving the 2 ϫ 6 transmembrane helix model. The same arrangement of transmembrane helices has been suggested in the case of P-glycoprotein (6, 7), and a large body of experimental data strongly favors this model (8, 9). On the other hand, Ling and co-workers (10, 11), by suggesting an alternative 6-and 4-helix conformation, raised the possibility that P-glycoprotein may exist in two different topological forms in the cell membrane.When the m...