RECONSTITUTION OF ATP-DEPENDENT LEUKOTRIENE C-4 TRANSPORT BY COEXPRESSION OF BOTH HALF-MOLECULES OF HUMAN MULTIDRUG-RESISTANCE PROTEIN IN INSECT CELLS

Multidrug resistance protein (MRP) confers a multidrug resistance phen otype similar to that associated with overexpression of P-glycoprotein . Unlike P-glycoprotein, MRP has also been shown to be a primary activ e ATP-dependent transporter of conjugated organic anions, The mechanis m(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown, To facilitate studies on the stru cture and function of MRP, we have determined whether a baculovirus ex pression system can be used to produce active protein, Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proxima l halves of the protein were expressed individually and in combination in Spodoptera frugiperda Sf21 cells, High levels of intact and half-l ength proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leuko triene C-4 (LTC(4)) with kinetic parameters very similar to those of M RP produced in transfected HeLa cells, Neither half-molecule was able to transport LTC(4). However, a functional transporter with characteri stics similar to those ofintact protein could be reconstituted vvhen b oth half-molecules were co expressed. Transport of LTC(4) by Sf21 memb rane vesicles containing either intact or reconstituted MRP was compet itively inhibited by both S-decylglutathione and 17 beta-estradiol 17- (beta-a-glucuronide), with K-i values similar to those reported previo usly for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Dee ley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682; L oe, 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 MR P produced in insect cells can function as an active transporter of LT C(4) and that the NH2- and COOH-proximal halves of the protein can ass emble efficiently to form a transporter with functional characteristic s similar to those of the intact protein.