ABCMdb

ABCC1 p.Gly1433Asp

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Publications

PMID: 15152943 [PubMed] Szentpetery Z et al: "Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants."

No. Sentence Comment

46 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
124 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 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 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 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 Nucleotide trapping of wild-type and G771D and G1433D signature mutant MRP1 variants. Login to comment
137 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 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 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 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
173 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
179 However, the G771D and G1433D signature mutants were practically inactive, both in the ATPase and in the vesicular transport assays. Login to comment
180 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 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

PMID: 15155846 [PubMed] Ren XQ et al: "Function of the ABC signature sequences in the human multidrug resistance protein 1."

No. Sentence Comment

3 We therefore investigated the effect of mutation of the signature sequences (G771D and G1433D) and of the Walker A motifs (K684M and K1333M) in the NBDs on the 8-azido-[␣-32 P]ATP photolabeling and 8-azido-[␣-32 P]ADP vanadate trapping of MRP1. Login to comment
6 The G1433D mutation in the signature motif of NBD2 enhanced the labeling of NBD2 but did not affect the labeling of NBD1. These effects were all substrate-independent. Login to comment
10 However, whereas the G1433D mutation also considerably inhibited trapping at NBD1, it only partially inhibited trapping of NBD2, and the trapping could still be enhanced by leukotriene C4. Login to comment
58 The strategies employed for site-directed mutagenesis of G771D and G1433D in MRP1 cDNA were described previously (Ren et al., 2001). Login to comment
59 The primer used to generate G771D and G1433D mutations were forward primers 5Ј-CTGGGGACCAGAAGCAGCGCGTGAG-3Ј and 5Ј-AGTGTCGATCAGCGCCAGCTTGTG-3Ј (underlining indicate mismatched bases encoding G771D and G1433D mutations, respectively). Login to comment
92 The expression levels of the N-terminal halves of the mutated dual NϩC fragments relative to that for wt NϩC were 0.98, 0.55, 0.41, 1.58, and 0.84 for N K684MϩC, NϩC K1333M, N K684MϩC K1333M, N G771DϩC, and NϩC G1433D, respectively. Login to comment
93 The expression levels of the C-terminal halves of the mutated dual NϩC fragments relative to that for wt NϩC were 0.85, 0.49, 0.37, 1.21, and 0.51 for N K684MϩC, NϩC K1333M, N K684MϩC K1333M, N G771DϩC ,and NϩC G1433D, respectively. Login to comment
116 Unlike the situation observed with the Walker A motifs, mutation of the signature sequence in the N (G771D) or the C (G1433D) - terminal half enhanced the labeling of the NBD in their respective fragments. Login to comment
129 However, whereas mutation of the signature sequence of NBD1 (G771D) completely inhibited the trapping at NBD2, mutation of the signature sequence of NBD2 (G1433D) only partially inhibited Fig. 5. Effect of NBD mutations on MRP1-ATP binding activity. Membrane vesicles (50 ␮g of protein) from insect cells coexpressing both N-and C-terminal wt or mutant fragments of MRP1 were incubated on ice with 5 ␮M 8-azido-[␣-32 P]ATP and 5 mM MgCl2 in the presence or absence of 1 ␮M LTC4 as described under Materials and Methods. Login to comment
141 Although ATP-dependent LTC4 transport by G771D and G1433D MRP1 mutants, as well as transport by the K684M and K1333M mutants in the Walker A motifs, were considerably decreased, GSH-dependent photolabeling with azido AG-A of these MRP1 mutants was retained. Login to comment
159 However, weak labeling of NBD2 and no trapping at NBD1 were observed when the G1433D mutant half molecule was coexpressed with the wild-type NH2-proximal half-molecule. Login to comment

PMID: 15252017 [PubMed] Szentpetery Z et al: "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."

No. Sentence Comment

2 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 The conserved glycines in the fourth position of the signature motifs (LSGGQ) were substituted for aspartic acids (G771D and G1433D). Login to comment
75 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 The trapping ability of the G771D and G1433D mutants was investigated under the same conditions as the wild type. Login to comment
89 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 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 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 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 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 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 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 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 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 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 q shows WT MRP1, f shows G771D, and ࡗ shows G1433D. Login to comment
184 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
194 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 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 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 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

PMID: 15355964 [PubMed] Zhao Q et al: "Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1."

No. Sentence Comment

15 The corresponding mutations in MRP1, G771D in NBD1 and G1433D in NBD2, almost completely abolished ATP-dependent LTC4 transport (20). Login to comment

PMID: 16861249 [PubMed] Ren XQ et al: "A functional role of intracellular loops of human multidrug resistance protein 1."

No. Sentence Comment

148 A previous study suggested that ATP binding to both NBDs of MRP1 was not abrogated when the signature sequence of NBD2 was mutated (G1433D) (14). Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

182 However, mutations of the conserved glycine residue at the fourth position of the signature sequence with aspartic acid to generate G771D in NBD1 or G1433D in NBD2 of MRP1 resulted in the loss of ability to hydrolyze ATP and to uptake solute [116]. Login to comment
183 Accordingly, the mutants at these positions, such as G771D, G771A, G1433D or G1433A, did not lose their ability to bind ATP, but significantly reduced their Vi-dependent nucleotide trapping at 37°C [61, 116, 117]. Login to comment
185 In contrast, the G1433D mutation in the signature sequence of NBD2 enhanced the [α-32 P]-ATP binding on ice at the mutated NBD2, but completely inhibited the Vi-dependent ADP trapping at the intact NBD1 at 37°C [118], implying that the LSGGQ signature sequence in NBD1 may be involved in ATP binding/hydrolysis at the NBD2, whereas the LSVGQ signature sequence in NBD2 may be involved in ATP binding/hydrolysis at the NBD1. Login to comment

PMID: 19949927 [PubMed] Chang XB et al: "Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1."

No. Sentence Comment

145 However, substitution of the conserved glycine residue at the fourth position of LSGGQ motif with an A or a D residue in NBD1 (G771D or G771A) or in NBD2 (G1433D or G1433A) lost their abilities to transport substrate across the membrane (99, 151, 152). Login to comment
147 Conversely, the G1433D mutation in the ABC signature sequence of NBD2 enhanced the (a-32 P)-ATP binding on ice at the mutated NBD2, but completely inhibited the Vi-dependent ADP trapping at the intact NBD1 at 37 ºC (153), implying that the LSGGQ signature motif in NBD1 may be involved in ATP binding at the NBD2, whereas the LSVGQ signature motif in NBD2 may be involved in ATP binding at the NBD1. Login to comment

PMID: 20842442 [PubMed] Long Y et al: "Molecular analysis and heavy metal detoxification of ABCC1/MRP1 in zebrafish."

No. Sentence Comment

142 Overexpression of ABCC1-G1420D sensitizes zebrafish embryos to heavy metals It has been shown that a mutation in the ABC signature of human ABCC1 (G771D or G1433D) abolished its ATP hydrolysis and transport function without effects on the substrates binding activity [26]. Login to comment