ALTERED DRUG-STIMULATED ATPASE ACTIVITY IN MUTANTS OF THE HUMAN MULTIDRUG-RESISTANCE PROTEIN

Citation
M. Muller et al., ALTERED DRUG-STIMULATED ATPASE ACTIVITY IN MUTANTS OF THE HUMAN MULTIDRUG-RESISTANCE PROTEIN, The Journal of biological chemistry, 271(4), 1996, pp. 1877-1883
Citations number
40
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
271
Issue
4
Year of publication
1996
Pages
1877 - 1883
Database
ISI
SICI code
0021-9258(1996)271:4<1877:ADAAIM>2.0.ZU;2-5
Abstract
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug ex trusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombina nt baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected ce lls the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-AT P binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domai ns, all these mutations abolished drug-stimulated ATPase activity inde pendent of the MgATP concentrations applied. Photoaffinity labeling wi th 8-azido-ATP showed that the double lysine mutant had a decreased AT P-binding affinity. In the MDR1 mutant containing a Gly(185) to Val re placement we found no significant alteration in the maximum activity o f the MDR1-ATPase or in its activation by verapamil and vinblastine, a nd this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly(185) to Val mutati on significantly increased the stimulation of the MDR1-ATPase by colch icine and etoposide, while slightly decreasing its stimulation by vinc ristine. These shifts closely correspond to the effects of this mutati on on the drug-resistance profile, as observed in tumor cells. These d ata indicate that the Sf9-baculovirus expression system for MDR1 provi des an efficient tool for examining structure-function relationships a nd molecular characteristics of this clinically important enzyme.