ABCMdb

ABCC1 p.Gly1433Asp

Predicted by SNAP2:	A: D (80%), C: D (80%), D: D (91%), E: D (95%), F: D (91%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (85%), P: D (91%), Q: D (91%), R: D (95%), S: D (80%), T: D (91%), V: D (91%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Functional studies on the MRP1 multidrug transport... Anticancer Res. 2004 Mar-Apr;24(2A):449-55. 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., [PMID:15152943]

Abstract [show]
BACKGROUND: MRP1 is a key multidrug resistance ATP-binding Cassette (ABC) transporter in tumor cells. A functionally important signature motif is conserved within all ABC domains. Our current studies aimed to elucidate the role of these motifs in the cooperation of MRP1 ABC domains. MATERIALS AND METHODS: We designed human MRP1 mutants based on a bacterial ABC structure. Conserved leucines (Leu) were replaced by arginines (Arg), while glycines (Gly) were substituted for aspartic acids (Asp). The activity of these mutants was assayed by measuring ATPase activity and vesicular transport. ATP-binding and transition-state formation were studied by a photoreactive ATP analog. RESULTS: The Leu to Arg mutants retained both ATPase and transport activity, while the Gly to Asp mutants were inactive in all functional assays, while showing normal ATP-binding. CONCLUSION: Our results reinforce the notion that a single mutation in one of the ABC-signature regions affects the function of the whole protein. The relative role of the conservative leucines and glycines in MRP1 indicates a similar three-dimensional structure within the catalytic center of various ABC proteins.

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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

[hide] Function of the ABC signature sequences in the hum... Mol Pharmacol. 2004 Jun;65(6):1536-42. Ren XQ, Furukawa T, Haraguchi M, Sumizawa T, Aoki S, Kobayashi M, Akiyama S
Function of the ABC signature sequences in the human multidrug resistance protein 1.
Mol Pharmacol. 2004 Jun;65(6):1536-42., [PMID:15155846]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is a membrane ATP-binding cassette transporter that confers multidrug resistance to tumor cells by effluxing intracellular drugs in an ATP-dependent manner. The mechanisms by which transport occurs and by which ATP hydrolysis is coupled to drug transport are not fully elucidated. In particular, the function of the signature sequences in the nucleotide binding domains (NBDs) of MRP1 is unknown. 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-[alpha-32P]ATP photolabeling and 8-azido-[alpha-32P]ADP vanadate trapping of MRP1. Both mutations in the Walker A motif almost completely inhibited the labeling of the mutated NBD with 8-azido-[alpha-32P]ATP but not the labeling of the other intact NBD. In contrast, the G771D mutation in the signature sequence of NBD1 enhanced the labeling of NBD1 but slightly decreased the labeling of NBD2. 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. Photolabeling of NBD2 and a very slight photolableing of NBD1 were detectable under vanadate trapping conditions with 8-azido-[alpha-32P]ATP. Trapping at both NBD1 and NBD2 was almost completely inhibited by K684M and K1333M mutations and by the K684M/K1333M double mutation. The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1. 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. Our findings suggest that both signature sequences of MRP1 are involved in ATP hydrolysis and must be intact for the ATP hydrolysis and the transport by MRP1.

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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

[hide] The role of the conserved glycines of ATP-binding ... J Biol Chem. 2004 Oct 1;279(40):41670-8. Epub 2004 Jul 12. 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 [PMID:15252017]

Abstract [show]
A key element of the structural model of ABC-ATP-ases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the gamma-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. 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 nucleotideas well as the transported substrate-protein interactions were studied. We found that these transport- and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide + oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.

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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

[hide] Mutation of the aromatic amino acid interacting wi... J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7. Zhao Q, Chang XB
Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1.
J Biol Chem. 2004 Nov 19;279(47):48505-12. Epub 2004 Sep 7., 2004-11-19 [PMID:15355964]

Abstract [show]
Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.

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

[hide] A functional role of intracellular loops of human ... J Biochem. 2006 Sep;140(3):313-8. Epub 2006 Jul 21. Ren XQ, Furukawa T, Yamamoto M, Aoki S, Kobayashi M, Nakagawa M, Akiyama S
A functional role of intracellular loops of human multidrug resistance protein 1.
J Biochem. 2006 Sep;140(3):313-8. Epub 2006 Jul 21., [PMID:16861249]

Abstract [show]
Multidrug resistance protein 1 (MRP1) is a human ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance to tumor cells by actively effluxing intracellular drugs. To examine the functional significance of intracellular loops (ICLs) in MRP1, we determined the effect of mutation of the amino acid sequence EXXXG, which is conserved in ICL5 and ICL7 of human MRP1, 2 and 3, sulfonylurea receptor (SUR) 1 and 2, and mouse MRP1 and 2. E and G in the ICLs of human MRP1 were mutated to L and P, respectively, and the N-terminal (including ICL5) and C-terminal (including ICL7) wild type or mutant halves of MRP1 were co-expressed in insect cells. The mutation of either ICL5 or ICL7 considerably decreased ATP-dependent LTC4 uptake into vesicles of insect cells expressing mutated MRP1. GSH-dependent photolabeling of MRP1 with an 125I-labeled photoaffinity analog of azido agosterol A (azido AG-A) was abolished by the mutations in ICL5 and ICL7. Mutations in ICL5 of MRP1 almost completely inhibited the labeling of NBD2, but not NBD1, by 8-azido-alpha-[32P]ATP. In contrast, mutations in ICL7 of MRP1 abolished the labeling of both NBDs. Mutation of either ICL5 or ICL7 of MRP1 almost completely inhibited vanadate trapping with 8-azido-alpha-[32P]ATP by both NBD1 and NBD2 domains. These findings indicate that the intramolecular signaling between NBD and ICLs in MRP1 is vital for MRP1 function.

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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

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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

[hide] Molecular analysis and heavy metal detoxification ... Mol Biol Rep. 2011 Mar;38(3):1703-11. Epub 2010 Sep 15. Long Y, Li Q, Cui Z
Molecular analysis and heavy metal detoxification of ABCC1/MRP1 in zebrafish.
Mol Biol Rep. 2011 Mar;38(3):1703-11. Epub 2010 Sep 15., [PMID:20842442]

Abstract [show]
ABCC1/MRP1 belongs to the ATP-binding cassette superfamily and its elevated expression is closely associated with the multidrug resistance of various tumor cells. In normal tissues, ABCC1 confers resistance to a wide variety of xenobiotics and toxicants, demonstrating its important roles in tissue defense. Here, we report the cloning and functional characterization of abcc1 gene in zebrafish. This gene is localized on zebrafish chromosome 3 and contains a 4,557 bp open-reading frame. The deduced polypeptide is composed of 1,518 amino acids, which shares 70% identity with human ABCC1. Phylogenetic analysis revealed that ABCC1 proteins from thirteen vertebrate species are highly conserved during evolution. Transcriptional expression of zebrafish abcc1 gene in developing embryos was examined by whole-mount in situ hybridization and real-time PCR. Transcripts of zebrafish abcc1 gene were detectable in four-cell stage embryos, indicating that this gene is maternally expressed. ABCC1 mRNAs were ubiquitously distributed in embryos before 12 h post-fertilization (hpf) and mainly localized in eyes and brain from 24 to 72 hpf, and in gills from 96 to 120 hpf. In addition, zebrafish abcc1 gene was highly expressed in 1-hpf embryos and detected in all adult tissues examined, with highest expression in testis and lowest in heart and liver. Exposure of ZF4 cells and embryos to CdCl(2).2.5H(2)O, HgCl(2), Pb(NO(3))(2), or Na(3)AsO(4).12H(2)O significantly induced transcriptional expression of abcc1 gene. Furthermore, overexpression of abcc1 improved the survival rates of embryos exposed to Cd, Hg or As, while overexpression of a abcc1 mutant (ABCC1-G1420D) sensitized zebrafish embryos to toxic metals. These data indicate that zebrafish ABCC1 has crucial roles in heavy metals detoxification.

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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