ABCMdb

PMID: 15252017

Szentpetery Z, Kern A, Liliom K, Sarkadi B, Varadi A, Bakos E
The role of the conserved glycines of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers.
J Biol Chem. 2004 Oct 1;279(40):41670-8. Epub 2004 Jul 12., 2004-10-01 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide- as well as the transported substrate-protein interactions were studied. Login to comment 49 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The conserved glycines in the fourth position of the signature motifs (LSGGQ) were substituted for aspartic acids (G771D and G1433D). Login to comment 75 ABCC1 p.Gly1433Asp ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp ABCC1 p.Gly771Asp In the transportand ATPase-incompetent mutants the conserved glycines in the fourth position of the LSGGQ motifs were substituted with glutamic acids (G771D and G1433D). Login to comment 85 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The trapping ability of the G771D and G1433D mutants was investigated under the same conditions as the wild type. Login to comment 89 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Collectively, these data indicate that in the G771D and G1433D signature mutants the MRP1*MgADP*anion transition state-like complex formation cannot be detected by azido-ATP labeling. Login to comment 108 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The half-maximal concentration of inhibition was 19.2 Ϯ 2.1 and 30.9 Ϯ 5.9 ␮M MgATP for G771D and G1433D, respectively. The differences in the apparent half-maximal effect of MgATP inhibition may be explained by lower affinity of the mutants toward MgATP. Login to comment 109 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Only a low level of cleavage product of G771D and G1433D mutants, resulting in the 85-kDa product, was observed in the FIG. 1. Login to comment 110 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Nucleotide trapping from [␣-32 P]8-azido-ATP of wild type (WT), G771D, and G1433D signature mutant MRP1 variants in isolated Sf9 membrane vesicles. Login to comment 129 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Deconvolution of the obtained bell-shaped curves revealed the following half-maximal concentration values: 0.10 Ϯ 0.01 and 4.68 Ϯ 0.44 ␮M MgATP for the rising and declining parts of the curve, respectively, in case of the cleavage of the wild type MRP1; 0.37 Ϯ 0.07 and 8.95 Ϯ 0.81 ␮M MgATP for the rising and for the declining parts of the curve, respectively, in the case of G771D; 0.41 Ϯ 0.07 and 10.15 Ϯ 0.96 ␮M MgATP for the rising and for the declining parts of the curve, respectively, in the case of G1433D. Login to comment 156 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp Cleavage reactions were performed at 37 °C; the MRP1 arrow indicates the full-length MRP1, and the f95 and f85 arrows indicate the cleavage products with molecular masses of 95 and 85 kDa, respectively. The positions of molecular-mass markers are indicated (kDa) at the right. A shows the immunoblots of cleavage reactions of WT (top gel), G771D (middle gel), and G1433D (bottom gel) performed in the presence of MgATP in concentrations as indicated below the immunoblots. B, densitometric evaluation of fragment f95 generated by cleavage reactions shown on A. Login to comment 158 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The plot shows the extent of cleavage resulting fragment f95; the plotted values were calculated by subtracting the relative densities from 100. q shows WT MRP1, f shows G771D, and ࡗ shows G1433D. Login to comment 161 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The data are expressed as relative density, with densities measured in the cleavage reaction in the presence of 500 ␮M MgATP arbitrarily set to 100. q shows WT MRP1, f shows G771D, and ࡗ shows G1433D. Login to comment 164 ABCC1 p.Gly1433Asp ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp ABCC1 p.Gly771Asp The declining part of the bell-shaped curves were shifted toward higher MgATP concentrations in the presence of etoposide ϩ GSSG in the case of both mutants (8.95 Ϯ 0.8 ␮M versus 12.8 Ϯ 1.4 ␮M MgATP in case of G771D and 10.15 Ϯ 0.9 ␮M versus 12.49 Ϯ 1.4 ␮M MgATP in case of G1433D), whereas no significant effect was observed in the presence of LTC4 in the MgATP concentration range (8.95 Ϯ 0.8 ␮M versus 10.0 Ϯ 0.9 ␮M MgATP in case of G771D and 10.15 Ϯ 1.0 ␮M versus 10.41 Ϯ 0.8 ␮M MgATP in case of G1433D). Login to comment 174 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The positions of molecular mass markers are indicated (kDa) at the right. A shows the immunoblots of cleavage reactions of WT (top gel), G771D (middle gel), and G1433D (bottom gel) performed in the presence of MgATP in concentrations as indicated below the immunoblots. B, densitometric evaluation of fragment f25 generated by cleavage reactions shown in A. Login to comment 177 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp q shows WT MRP1, f shows G771D, and ࡗ shows G1433D. Login to comment 184 ABCC1 p.Leu768Arg ABCC1 p.Gly1433Asp ABCC1 p.Leu1430Arg ABCC1 p.Gly771Asp These mutations affect the conserved LSGGQ motifs in either ABC domain; the leucines were replaced with arginines (L768R and L1430R), and the glycines in position 4 were substituted with glutamic acids (G771D and G1433D). Login to comment 185 ABCC1 p.Leu3Arg It was found that the mutants could be expressed in Sf9 insect cells as effectively as the wild type MRP1, and the Leu 3 Arg replacements do not effect the drug-stimulated ATPase activity or the ATP-dependent LTC4 transport activity of the MRP1-variants. Login to comment 188 ABCC1 p.Gly771Asp It was concluded that the conserved glycine residues in the fourth position of the LSGGQ motifs are essential in MRP1-related function.2 It is worthwhile to note that the conservation of the glycine residue in the fourth position of the ABC signature region is universal in the ABC family and that the MRP1 G771D mutation is analogous to a disease-causing mutation in (G551D), which is associated with a severe form of cystic fibrosis. Login to comment 194 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp A, vanadate-induced cleavage reactions of wild type (top gel), G771D (middle gel), and G1433D (bottom gel) in the presence of etoposide (0.5 mM) ϩ GSSG (2 mM) and LTC4 (0.8 ␮M), respectively. The reaction was performed at 37 °C at 2 ␮M MgATP concentration as indicated below the immunoblots. Login to comment 200 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp As is shown in Fig. 1, neither G771D nor G1433D could perform nucleotide trapping irrespective of which inhibitory anion was present. Login to comment 202 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp These data indicate that in the G771D and G1433D signature mutants the MRP1*- MgADP*anion transition state-like complex formation cannot be detected by azido-ATP labeling. Login to comment 211 ABCC1 p.Gly1433Asp ABCC1 p.Gly771Asp The differences in the apparent half-maximal effect of MgATP inhibition on cleavage at site I (5.6 Ϯ 1.0 ␮M MgATP in case of wild type versus 19.2 Ϯ 2.1 and 30.9 Ϯ 5.9 ␮M MgATP, for G771D and G1433D, respectively) may be explained by the lower affinity of the mutants toward MgATP (Fig. 3B). Login to comment