ABCMdb

PMID: 11336637

Szakacs G, Ozvegy C, Bakos E, Sarkadi B, Varadi A
Role of glycine-534 and glycine-1179 of human multidrug resistance protein (MDR1) in drug-mediated control of ATP hydrolysis.
Biochem J. 2001 May 15;356(Pt 1):71-5., 2001-05-15 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D\G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes. Login to comment 43 ABCB1 p.Gly534Asp MATERIALS AND METHODS Construction of the recombinant transfer vectors The recombinant baculovirus transfer vector carrying the G534D-MDR1 variant was constructed previously [19]. Login to comment 48 ABCB1 p.Gly1179Asp Two internal complementary primers were used, each containing the specific mutation (G1179D, 5h-TCTGGTGATCAGAAACAACGCAT-3h). Login to comment 57 ABCB1 p.Gly1179Asp The product and the pAcUW21-LMDR1 vector were digested with NotI and PstI, and the product was ligated into the vector, resulting in pAcUW21-L-G1179D. Login to comment 59 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Asp The pAcUW21-G534D\G1179D construct was engineered by replacing the 1177-3372 EcoRI-PstI fragment of pAcUW21-G534D with that of pAcUW21-L- G1179D. Login to comment 79 ABCB1 p.Gly534Val ABCB1 p.Gly534Asp In the same study we found that the MDR1 variant G534D was expressed at a level comparable with the wild-type protein, whereas the expression of a similar variant (G534V-MDR1) was not detected. Login to comment 80 ABCB1 p.Gly534Val In the present study, with careful selection and subsequent amplification of individual clones, we were able to express high levels of the G534V mutant as well (Figure 1). Login to comment 81 ABCB1 p.Gly1179Asp ABCB1 p.Gly1179Asp ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp ABCB1 p.Gly534Asp Figure 1 also shows that both the G1179D mutant (affecting the equipositional glycine in the C-terminal ABC unit) and the variant containing aspartic residues at both sides (G534D\ Figure 1 Expression of the MDR1 signature mutants Isolated Sf9 cell membranes (10 µg) expressing G534D-MDR1 (lane 1), G1179D-MDR1 (lane 2), G534D/G1179D-MDR1 (lane 3), G534V-MDR1 (lane 4) or wild-type MDR1 (lane 5) were run on SDS/7.5% (w/v) PAGE gels. Login to comment 85 ABCB1 p.Gly1179Asp G1179D) could be expressed efficiently in Sf9 cells. Login to comment 96 ABCB1 p.Gly1179Asp ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp ABCB1 p.Gly534Asp ABCB1 p.Gly534Asp We observed nucleotide trapping in mutants G534D, Figure 2 Binding of [α-32 P]8-azido-ATP by the MDR1 signature mutants ATP-binding was performed with isolated Sf9 cell membranes (100 µg) expressing G534V-MDR1 (lane 1), wild-type MDR1 (lanes 2 and 8), G534D-MDR1 (lanes 3 and 4), β- galactosidase (lane 5), G534D/G1179D-MDR1 (lane 6) or G1179D-MDR1 (lane 7). Login to comment 100 ABCB1 p.Gly1179Asp ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp ABCB1 p.Gly534Asp Membranes expressing G534D/G1179D-MDR1 (lanes 1 and 2), G534V-MDR1 (lanes 3-5), G534D-MDR1 (lanes 6-8) or G1179D-MDR1 (lanes 9-11) were incubated at 37 mC for 10 min in the presence of 20 µM [α-32 P]8-azido-Mg-ATP as described in the Materials and methods section. Login to comment 104 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val G534V and G1179D in the presence of AlF % - (see Figure 3) or BeFx (results not shown). Login to comment 107 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp Interestingly, neither G534D-MDR1 nor G1179D-MDR1 was labelled in the presence of vanadate, not even at higher azido-ATP concentrations and longer incubation times (up to 100 µM and 10 min respectively), whereas G534V showed vanadate-induced nucleotide trapping activity (results not shown). Login to comment 114 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp Conversely, the same reaction with the ABC signature mutants G534D (ratio 0.32), G534V (ratio 0.41) and G1179D (ratio 0.48) was inhibited by verapamil (36 µM). Login to comment 116 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp Inhibition persisted over a wide range of azido-ATP concentration (5-50 µM) and was observed in the presence of other drugs (calcein-AM, vincristine, cyclosporin A; results not Figure 4 Effect of verapamil on transition-state formation (nucleotide trapping) in the presence of AlF4 - Labelling was performed with isolated Sf9 cell membranes (100 µg) expressing wild-type MDR1 (lanes 1 and 2), G534V-MDR1 (lanes 3 and 4), G534D-MDR1 (lanes 5 and 6) or G1179D-MDR1 (lanes 7 and 8) as described in the Materials and methods section. Login to comment 119 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp The positions of molecular-mass markers are indicated (in kDa) at the right. Figure 5 Limited proteolysis of the ABC signature mutants after nucleotide trapping in the presence of AlF4 - Labelling was performed with isolated Sf9 cell membranes (200 µg) expressing wild-type MDR1 (lane 1), G534D-MDR1 (lane 2), G534V-MDR1 (lane 3), G1179D-MDR1 (lane 4) or β- galactosidase (lane 5). Login to comment 130 ABCB1 p.Gly1179Asp ABCB1 p.Gly534Val ABCB1 p.Gly534Asp Nevertheless, as shown in Figure 5, catalytic intermediates stabilized by AlF % - were formed in both ABC domains of the G534D, G534V and the G1179D mutants (BeFx gave similar results; results not shown). Login to comment