ABCMdb

PMID: 15152943

Szentpetery Z, Sarkadi B, Bakos E, Varadi A
Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants.
Anticancer Res. 2004 Mar-Apr;24(2A):449-55., [PubMed]

Sentences

No. Mutations Sentence Comment

35 ABCC1 p.Trp1246Cys Substitution of a highly conserved tryptophan at position 1246 with cysteine in TM17 was also shown to modulate the substrate specificity of MRP1 (21). Login to comment 42 ABCC1 p.Lys1333Met ABCC1 p.Lys684Met When a highly conserved lysine residue within the Walker A motif in MRP1 was substituted for methionine in either the N-ABC or in the C-ABC unit, the K1333M mutation in the C-ABC nearly abolished ATP-dependent LTC4 uptake, whereas the K684M substitution in the N-ABC had a less pronounced effect (22,23). Login to comment 46 ABCC1 p.Leu768Arg ABCC1 p.Gly1433Asp ABCC1 p.Leu1430Arg ABCC1 p.Gly771Asp The conserved leucines of the LSGGQ motifs were replaced by arginines (L768R, L1430R) and the conserved glycines in the fourth position of the signature motifs were substituted for aspartic acids (G771D, G1433D). Login to comment 122 ABCC1 p.Leu768Arg ABCC1 p.Leu1430Arg In Figure 2 we document that, when measuring the ATPase activity of the L768R and L1430R signature mutants, we found that they possessed a significant level of vanadate-sensitive, drug-stimulated ATPase activity. Login to comment 124 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp On the other hand, in the G771D and G1433D mutants the NEM-GS- stimulated ATPase activities were not significantly higher than those in the negative control ‚-galactosidase-infected cell membranes (5.8 and 4.5 nmol Pi/mg membrane protein/min, respectively). Login to comment 128 ABCC1 p.Leu768Arg ABCC1 p.Gly1433Asp ABCC1 p.Leu1430Arg ABCC1 p.Gly771Asp As shown in Figure 3, in harmony with the ATPase activity measurements, the L768R and L1430R signature mutants presented 87-89 % of the transport activity of the wild-type MRP1, while the G771D and G1433D signature mutants had only a negligible level of ATP-dependent LTC4 uptake, similar to that found in the ‚-galactosidase- expressing control membranes. Login to comment 129 ABCC1 p.Leu768Arg ABCC1 p.Gly1433Asp ABCC1 p.Leu1430Arg ABCC1 p.Gly771Asp All these functional studies indicated that the catalytic cycles of the L768R and L1430R mutants were similar to the wild-type, while that of the G771D and G1433D mutants were seriously diminished. Login to comment 131 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp In order to examine whether the loss of ATPase and transport activity in the G771D and G1433D signature mutants was due to an impaired binding of ATP, we performed photoaffinity-labeling experiments using [·- 32P]8-azido-ATP under nonhydrolytic conditions. Login to comment 133 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Nucleotide trapping of wild-type and G771D and G1433D signature mutant MRP1 variants. Login to comment 137 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Sf9 membranes containing the wild-type and the G771D and G1433D signature MRP1 variants were incubated on ice in the presence of 5 ÌM [·-32P]8-azido-ATP. Login to comment 148 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp In experiments documented in Figure 5, isolated Sf9 membranes containing the wild-type MRP1, or the G771D and G1433D signature mutants, were incubated under hydrolytic conditions for 5 min, in the presence of vanadate as an inhibitory anion, and 5 ÌM [·-32P]8-azido-ATP. Login to comment 150 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp As shown, the G771D and G1433D mutants, although displaying significant 8-azido-ATP-binding (see above), did not perform any nucleotide trapping. Login to comment 154 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Collectively these data indicate that the G771D and G1433D signature mutants are capable of proper ATP-binding, but a later step of the catalytic cycle, namely the transition state formation, cannot be detected. Login to comment 172 ABCC1 p.Leu768Arg ABCC1 p.Leu1430Arg We chose leucine, a residue not oriented towards the ATP, which we substituted for arginine (L768R, L1430R). Login to comment 173 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The glycines of the fourth position of the signature motif, laying on the ATP-binding surface, were replaced by aspartic acids (G771D, G1433D). Login to comment 178 ABCC1 p.Leu768Arg ABCC1 p.Leu1430Arg We found that the ATPase and transport activity of the L768R and L1430R signature mutants, reflecting the whole catalytic cycle of MRP1, were not significantly different from that of the wild-type MRP1. Login to comment 179 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp However, the G771D and G1433D signature mutants were practically inactive, both in the ATPase and in the vesicular transport assays. Login to comment 180 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp In order to examine whether the loss of ATPase activity in the G771D and G1433D signature mutants was due to an impaired binding of ATP, we performed photoaffinity-labeling experiments. Login to comment 183 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp When studying the formation of a transition state of the ATPase cycle, reflected by vanadate-dependent nucleotide trapping, we found that in the G771D and G1433D mutants this partial reaction could not be detected. Login to comment