Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region

Details

Serval ID
serval:BIB_EDCB34315A03
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region
Journal
Biochemical Journal
Author(s)
Bakos  E., Klein  I., Welker  E., Szabo  K., Muller  M., Sarkadi  B., Varadi  A.
ISSN
0264-6021 (Print)
Publication state
Published
Issued date
05/1997
Volume
323
Number
3
Pages
777-783
Notes
Comparative Study Journal Article Research Support, Non-U.S. Gov't --- Old month value: May 1
Abstract
A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.
Keywords
ATP-Binding Cassette Transporters/chemistry/*genetics Adenosine Triphosphate/*metabolism Amino Acid Sequence Animals Binding Sites Calcium Channel Blockers/pharmacology Consensus Sequence DNA, Complementary/genetics Drug Resistance, Multiple/genetics Fluoresceins/pharmacology Genetic Vectors Humans Kinetics Models, Molecular Mutagenesis, Site-Directed Nucleopolyhedrovirus/genetics P-Glycoprotein/chemistry/*genetics Recombinant Fusion Proteins/metabolism Rhodamine 123 Rhodamines/pharmacology Spodoptera/cytology Structure-Activity Relationship Valinomycin/pharmacology Verapamil/pharmacology
Pubmed
Web of science
Create date
24/01/2008 14:40
Last modification date
20/08/2019 16:15
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