Douglas W. Loe

Multidrug Resistance Protein (MRP)-mediated Transport of Leukotriene C and Chemotherapeutic Agents in Membrane Vesicles DEMONSTRATION OF GLUTATHIONE-DEPENDENT VINCRISTINE TRANSPORT

The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C (LTC) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V and K values for LTC transport by membrane vesicles from T14 cells were 529 ± 176 pmol mg min and 105 ± 31 nM, respectively. At 50 nM LTC, the K (ATP) was 70 μM. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC transport (K 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC transport and prevents photolabeling of MRP by LTC, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent.

Reconstitution of ATP-dependent Leukotriene C4 Transport by Co-expression of Both Half-molecules of Human Multidrug Resistance Protein in Insect Cells

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein. Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proximal halves of the protein were expressed individually and in combination in Spodoptera frugiperda Sf21 cells. High levels of intact and half-length proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leukotriene C4 (LTC4) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells. Neither half-molecule was able to transport LTC4. However, a functional transporter with characteristics similar to those of intact protein could be reconstituted when both half-molecules were co-expressed. Transport of LTC4 by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17β-estradiol 17-(β-D-glucuronide), with Ki values similar to those reported previously for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682; Loe, D. W., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689). These studies demonstrate that human MRP produced in insect cells can function as an active transporter of LTC4 and that the NH2- and COOH-proximal halves of the protein can assemble efficiently to form a transporter with functional characteristics similar to those of the intact protein.

Multidrug resistance protein. Identification of regions required for active transport of leukotriene C4.

Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4). We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC4 transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.