Abstract
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 17beta-estradiol 17-(beta-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.
Highlights
Of drugs that includes many natural products and their derivatives
The effects of S-decylglutathione and 17-estradiol 17-(-D-glucuronide) on leukotriene C4 (LTC4) uptake by Sf21 vesicles prepared from cells infected with vectors expressing either intact multidrug resistant protein (MRP) or both half-molecules were determined at various LTC4 concentrations in the presence of a fixed concentration of inhibitor
Generation of Recombinant Baculoviruses—Fig. 1 illustrates a possible topology of MRP, showing the maximal number of transmembrane regions predicted from hydropathy analyses [5, 30]
Summary
P-gp, P-glycoprotein; MRP, multidrug resistance protein; LTC4, leukotriene C4; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; NBD, nucleotide binding domain; CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP binding cassette; PNGase F, peptide N-glycosidase F; wt, wild type. We found that neither half-molecule alone would support LTC4 transport, but a high level of ATP-dependent transport was obtained when both half-molecules were co-expressed. These results indicate that the two half-molecules of MRP can assemble to form an active transporter despite the lack of covalent linkage, and that transport of LTC4 requires interaction of both halves of MRP
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