Gao M, Loe D W, Grant C E, Cole S P, Deeley R G
Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biol Chem. 1996 Nov 1;271(44):27782-7. doi: 10.1074/jbc.271.44.27782.
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.
多药耐药蛋白（MRP）赋予的多药耐药表型与P-糖蛋白过表达相关的表型相似。与P-糖蛋白不同，MRP还被证明是一种依赖ATP的共轭有机阴离子的原发性主动转运蛋白。MRP转运这些化合物并增加对天然产物药物耐药性的机制尚不清楚。为了便于对MRP的结构和功能进行研究，我们确定了杆状病毒表达系统是否可用于产生活性蛋白。全长MRP以及与该蛋白的NH2端或COOH端近端一半相对应的分子分别或组合在草地贪夜蛾Sf21细胞中表达。在感染细胞的膜泡中检测到高水平的完整和半长蛋白。尽管糖基化不足，但全长蛋白转运白三烯C4（LTC4）的动力学参数与转染的HeLa细胞中产生的MRP非常相似。两个半分子均不能转运LTC4。然而，当两个半分子共表达时，可以重建一个具有与完整蛋白相似特征的功能性转运蛋白。含有完整或重组MRP的Sf21膜泡对LTC4的转运受到S-癸基谷胱甘肽和17β-雌二醇17-（β-D-葡萄糖醛酸）的竞争性抑制，其Ki值与先前报道的HeLa细胞中表达的MRP相似（Loe，D.W.，Almquist，K.C.，Deeley，R.G.和Cole，S.P.C.（1996）J.Biol.Chem.271，9675-9682；Loe，D.W.，Almquist，K.C.，Cole，S.P.C.和Deeley，R.G.（1996）J.Biol.Chem.271，9683-9689）。这些研究表明，在昆虫细胞中产生的人MRP可以作为LTC4的活性转运蛋白发挥作用，并且该蛋白的NH2端和COOH端近端一半可以有效组装形成具有与完整蛋白相似功能特征的转运蛋白。
