FUNCTIONAL-CHARACTERIZATION OF A GLYCINE 185-TO-VALINE SUBSTITUTION IN HUMAN P-GLYCOPROTEIN BY USING A VACCINIA-BASED TRANSIENT EXPRESSION SYSTEM

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that c onfers multidrug resistance to otherwise sensitive cells. A mutation i n Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines t hat are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutat ion leads to an altered drug resistance profile, we used a transient e xpression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of m any aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human oste osarcoma (HOS) cells by infection with a recombinant vaccinia virus en coding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant an d wild-type proteins, photolabeling with [H-3]azidopine and [I-125]iod oarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodami ne 123, and accumulation of radiolabeled vinblastine and colchicine we re evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant t han for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosp orin A inhibited the ATPase activity of the mutant more efficiently th an that of the wild-type. This latter observation explains the enhance d potency of cyclosporin A as an inhibitor of the mutant Pgp. No diffe rences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblast ine in cells expressing the mutant protein, as compared with those exp ressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase acti vity and altered interaction with substrates and the inhibitor cyclosp orin A.