Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein

Details

Serval ID
serval:BIB_E86D1A673246
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein
Journal
Journal of Biological Chemistry
Author(s)
Muller  M., Bakos  E., Welker  E., Varadi  A., Germann  U. A., Gottesman  M. M., Morse  B. S., Roninson  I. B., Sarkadi  B.
ISSN
0021-9258 (Print)
Publication state
Published
Issued date
01/1996
Volume
271
Number
4
Pages
1877-83
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Jan 26
Abstract
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion 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 recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
Keywords
Adenosine Triphosphatases/chemistry/*metabolism Adenosine Triphosphate/*metabolism Affinity Labels Animals Base Sequence Cell Membrane/metabolism DNA Primers/chemistry Humans Molecular Sequence Data Mutagenesis, Site-Directed P-Glycoprotein/chemistry/*metabolism Spodoptera Structure-Activity Relationship Vanadates/pharmacology
Pubmed
Web of science
Create date
24/01/2008 14:40
Last modification date
20/08/2019 16:11
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