This project seeks to examine the role of gp150-180 in multidrug resistance (MDR) and its role in the circumvention of MDR by calcium channel blockers. A major 150-180 kDa Vinca alkaloid (gp150-180) was identified in purified plasma membranes from MDR cells by photoaffinity labeling. Gp150-180 exhibits cross binding specificity for Vinca alkaloids, actinomycin D, anthracycline antibiotics, calcium channel blockers, and calmodulin inhibitors suggesting a central functional role in MDR phenotype and its circumvention. The role of gp150-180 in MDR will be tested by examining the effect of specific drug photolabeling on cellular drug uptake and efflux, and by drug uptake and release using membrane vesicles from MDR cells. the role of this drug acceptor protein in circumventing MDR will be examined by competition experiments using agents previously shown to counteract multidrug resistance. The subcellular distribution of gp150-180 over time will be monitored. For further characterization of drug binding sites and exploring whether gp150-180 possesses multiple drug binding sites, or whether it may be a family of proteins, each with specificity for a distinct class of drugs, the protein will be purified using drug affinity chromatographic methods. The purified gp150-180 will be inserted into planar lipid bilayers for functional reconstitution experiments and analyzing the role of this protein in cellular drug accumulation. The purified gp150-180 will be used to prepare monoclonal antibodies to define plasma membrane orientation and drug binding sites, and to evaluate the possible microheterogeneity of gp150-180. Detailed characterization of drug binding sites of gp150-180 may elucidate some of the underlying mechanisms of multidrug resistance and might lead to the development of new compounds designed to prevent or reverse the development of the multidrug resistant phenotype.
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