ABC transporters such as P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1), and the mitoxantrone-resistance protein (MXR, also known as breast cancer resistance protein, BCRP, or ABCP), which function as an ATP-dependent efflux pumps, play an important role in the development of multidrug resistance in most cancers. In addition, some of the other members of MRP subfamily (MRP2-5) also transport anticancer agents in a conjugated form. Thus, these transporters also may contribute to the development of multidrug resistance in malignant cells. Multidrug resistance-linked ABC transporters can recognize and transport a wide variety of amphipathic cytotoxic natural product anticancer drugs. Our studies are directed toward understanding the mechanism of action of the multidrug resistance-linked ABC transporters such as P-gp and MRP1. Recent studies with P-gp deal with the interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified P-gp suggest that ADP release is the rate-limiting step in the catalytic cycle, and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of P-gp, one in the transport of drug and the other in effecting con-formational changes to reset the transporter for the next catalytic cycle. We have further exploited the vanadate (Vi)-induced ADP trapped transition-state conformation of P-gp to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a decrease in the affinity of nucleotide (ATP or ADP) for P-gp coincident with the impaired substrate binding. The kinetics of repeating succession of trapping and release of [a-32P]-8-azidoADP through an entire catalytic cycle was determined, and we also monitored the substrate binding at the beginning and end of each trapping event. Though the two hydrolysis events have different functional outcomes vis-a-vis the recovery of substrate binding and translocation, they show comparable kinetic properties for both incorporation and release of nucleotide and the Km for [a-32P]-8-azidoATP in the presence of vanadate is identical. These data demonstrate that both nucleotide-binding domains behave symmetrically, and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time and the conformational change in this site that drastically decreases (>30-fold) the affinity of the second site for the ATP-binding. Thus, the blocking of ATP binding to the second site, while the first one is in catalytic conformation, appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by P-gp. Analyses of thermodynamic parameters indicate that 100-115 kJ/mole energy of activation is required for the drug (substrate)-stimulated ATP hydrolysis by P-gp. We demonstrated that the properties of the transition state intermediate of P-gp generated in the absence or presence of ATP hydrolysis is functionally indistinguishable. However, the trapping of P-gp with ADP in the absence of hydrolysis requires ~2.5-fold higher energy of activation compared with that observed when the transition state intermediate is generated through hydrolysis of ATP. Another unique feature of catalytic cycle of ATP hydrolysis by P-gp is that the substrates that stimulate steady-state ATP hydrolysis as well as the formation of transition state through hydrolysis of ATP, inhibit the formation of transition state intermediate in the absence of ATP hydrolysis. Thus the substrate-stimulated hydrolysis of ATP by P-gp appears to be a vectorial process, and this is consistent with its physiological role in drug transport. The multidrug resistance protein (MRP1) similar to P-gp plays an important role in the development of multidrug resistance in cancer cells. To investigate the interdomain interactions of the transporter molecule, we constructed numerous recombinant variants of wild-type MRP1 by mutating the glycosylation sites and inserting the Flag epitope sequence in different extracellular positions. We detected the accessibility of the Flag-tag in the different constructs by M2 monoclonal antibody and found that the accessibility of the epitope was dependent on the utilization of the glycosylation sites. The Flag epitope was accessible in unglycosylated MRP1, whereas the epitope was completely masked in the partially or fully-glycosylated protein. The Flag-tagged unglycosylated MRP1 variants along with the wild-type protein were stably expressed in both HEK293 and HeLa cells. The flag-tagged MRP1 stable transfectants exhibit drug (daunorubicin, vinblastine and VP-16) resistance profile similar to those expressing wild-type MRP1 protein. In addition, we have selected stable transfectants, which overexpress flag-tagged MRP1 variants by selection with step-wise increase in the concentration of VP-16. These highly resistant transfectants will be used for the structure-function analysis of MRP1.
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