Our efforts are directed toward understanding the mechanism of action of the multidrug resistance linked P-glycoprotein (P-gp) or the multidrug transporter. P-gp, a product of the MDR1 gene in humans, is a member of the ATP-Binding Cassette (ABC) superfamily of transporters. P-gp functions as an ATP-dependent efflux pump for a variety of hydrophobic agents including cytotoxic natural product anticancer drugs, reversing agents and peptides. Our major goals include the determination of the interaction between the substrate-binding domain(s), the ATP binding/utilization sites of the transporter, and also the role of ATP hydrolysis in drug translocation by P-gp through the cell membrane. We are employing biochemical and molecular biological approaches for the development of model systems such as an in vitro artificial membrane system and the heterologous baculovirus-insect cell expression system for the analysis of wild- type and mutant P-gps. We have developed procedures for large scale purification of recombinant P-gp with full retention of biological activity from a baculovirus expression system. To facilitate the structural studies, we have expressed the glycosylation-deficient P-gp in the insect cell system and the mutant P-gp retains drug transport function similar to the wild-type protein. The analysis of effect of various anticancer drugs as well as reversing agents and peptides on the ATPase activity indicates that the ATP hydrolysis of P-gp is modulated by interaction between overlapping catalytic and inhibitory sites. We have used an antibody- depletion method to determine the number of P-gp molecules present at the cell surface of a variety of multidrug resistant cells, and this information was used to determine the stoichiometry of vinblastine transport and vinblastine-stimulated ATP hydrolysis. Our results demonstrate that there is a tight coupling between drug transport and ATP hydrolysis and that hydrolysis of 2-3 molecules of ATP is required to transport one molecule of vinblastine. Using highly efficient photoaffinity labeling of P-gp with a prazosin analog, we have presented evidence for the presence of two non-identical drug binding sites on the multidrug transporter. In addition, drug binding to P-gp appears to be modulated by agents that bind to a modulator site that is separate from the substrate binding site.
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