Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.

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LRP is involved in resistance to doxorubicin, vincristine, etoposide, paclitaxel, and gramicidin D and has an important role in the transport of doxorubicin from the nucleus to the cytoplasm.

Inhibition of dThdPase in human colorectal carcinomas might improve prognosis for some patients.

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MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD 0 ), which is connected to a P-glycoprotein-like core region (⌬MRP) by a cytoplasmic linker domain zero (L 0 ).It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD 0 and L 0 are not known. We synthesized [125 I]11-azidophenyl agosterol A ([ 125 I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C 932-1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C 4 . Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L 0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L 0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (Pgp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1-4). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5), they share only 15% amino acid sequence identity (6). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD 0 ), which is connected to the P-gp-like core (⌬MRP) by a cytoplasmic linker domain zero (L 0 ) (7, 8).The precise roles of TMD 0 and L 0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9, 10), there is compelling evidence that they function very differently in drug transport (6, 11). P-gp confers multidrug resistance by directly binding and transporting unmodified drugs (12, 13). MRP1, however, is an active transporter of amphiphilic conjugated organic anions, including a number of compounds conjugated with GSH, glucuronide, and sulfate. To date, leukotriene C 4 (LTC 4 ) is the best substrate for MRP1 (14). It has been reported that GSH plays a critical role in MRP1-mediated MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine su...

The canalicular multispecific organic anion transporter (cMOAT), also termed MRP2, is a recently identified ATP-binding cassette transporter. We previously established stable human cMOAT cDNA-transfected cells, LLC/cMOAT-1 from LLC-PK1 cells, and LLC/CMV cells that were transfected with an empty vector. We found that LLC/cMOAT-1 cells have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxy-camptothecin, and cisplatin but not to etoposide. The multidrug resistance-reversing agents cyclosporin A (CsA) and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) almost completely reversed the resistance to VCR, 7-ethyl-10-hydroxy-camptothecin, and cisplatin of LLC/cMOAT-1 cells; and DL-buthionine-(S,R)-sulfoximine, (3'-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833), and 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-((3-dimethylamino-3- oxopropyl)-thio)-methyl]thio)propanoic acid (MK571) partially reversed the resistance to these drugs. CsA and PAK-104P at 10 microM enhanced the accumulation of VCR in LLC/cMOAT-1 cells almost to the level in LLC/CMV cells without the agents. The efflux of VCR from LLC/cMOAT-1 cells was enhanced compared with LLC/CMV cells and inhibited by CsA and PAK-104P. Transport of leukotriene C(4) (LTC(4)) and S-(2, 4-dinitrophenyl)glutathione also was studied with membrane vesicles prepared from these cells. LTC(4) and S-(2, 4-dinitrophenyl)glutathione were actively transported into membrane vesicles prepared from LLC/cMOAT-1 cells. The K(m) and V(max) values for the uptake of LTC(4) by the LLC/cMOAT-1 membrane vesicles were 0. 26 +/- 0.05 microM and 7.48 +/- 0.67 pmol/min/mg protein, respectively. LTC(4) transport was competitively inhibited by PAK-104P, CsA, MK571, and PSC833, with K(i) values of 3.7, 4.7, 13.1, and 28.9 microM, respectively. These findings demonstrate that cMOAT confers a novel drug-resistance phenotype. CsA and PAK-104P may be useful for reversing cMOAT-mediated drug resistance in tumors.