Our efforts are directed toward understanding the mechanism of action of the multidrug resistance linked P-glycoprotein (P-gp). P-gp also known as the multidrug transporter, 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) and 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 vaccinia virus based in vivo expression for the analysis of wild type and mutant P-gps. By employing site-directed mutagenesis to substitute all phosphorylation sites in the linker region of the protein we have demonstrated that the phosphorylation of P-gp is not essential for its ability to confer drug resistance phenotype to otherwise sensitive cells. 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. In addition, we have also initiated studies to identify the drug binding site(s) of human P-gp. These studies are progressing rapidly mainly due to the development of procedures for large scale purification of recombinant P-gp from a baculovirus expression system and increased efficiency of photoaffinity labeling of protein with drug analogs.
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