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
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.