title: Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
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.
authors.affiliation: National Institute of Haematology, Blood Transfusion and Immunology, Budapest, Hungary.
authors.forename: M
authors.initials: M
authors.lastname: Müller
authors.forename: E
authors.initials: E
authors.lastname: Bakos
authors.forename: E
authors.initials: E
authors.lastname: Welker
authors.forename: A
authors.initials: A
authors.lastname: Váradi
authors.forename: U A
authors.initials: UA
authors.lastname: Germann
authors.forename: M M
authors.initials: MM
authors.lastname: Gottesman
authors.forename: B S
authors.initials: BS
authors.lastname: Morse
authors.forename: I B
authors.initials: IB
authors.lastname: Roninson
authors.forename: B
authors.initials: B
authors.lastname: Sarkadi
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014638:Vanadates / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D017931:DNA Primers; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases