Reconstitution of ATP-dependent leukotriene C-4 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 C-4 (LTC(4)) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells, Neither half-molecule was able to transport LTC(4). However, a functional transporter with characteristics similar to those ofintact protein could be reconstituted vvhen both half-molecules were co expressed. Transport of LTC(4) by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17 beta-estradiol 17-(beta-a-glucuronide), with K-i 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 LTC(4) 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.