ABCMdb

ABCC1 p.Asp1454Asn

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

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Functional interactions between nucleotide binding... Mol Pharmacol. 2005 Jun;67(6):1944-53. Epub 2005 Mar 8. Payen L, Gao M, Westlake C, Theis A, Cole SP, Deeley RG
Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1).
Mol Pharmacol. 2005 Jun;67(6):1944-53. Epub 2005 Mar 8., [PMID:15755910]

Abstract [show]
Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH(2)-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C(4) (LTC(4))(.) Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (beta,gamma-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) in decreasing LTC(4) photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPgammaS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC(4) binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the gamma-phosphate of the bound nucleotide.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
67 The forward primers for the D792N and D1454N mutations of the Walker B motifs were 5Ј-CATTTACCTCT- TCAATGATCCCCTC-3Ј and 5Ј-ATCCTTGTGTTGAATGAGGCCA- CG-3Ј, respectively. Login to comment
189 The D792N mutation diminished transport activity by approximately 65%, whereas the D1454N mutation essentially inactivated the protein (Fig. 5B). Login to comment
191 In contrast, the D1454N mutation eliminated photolabeling of only NBD2 and had no effect on photolabeling of NBD1 (Fig. 5C). Login to comment
192 The D792N mutation also strongly decreased ADP trapping by both NBD1 and NBD2, although the D1454N mutation eliminated trapping by NBD2 but only modestly decreased trapping by NBD1 (Fig. 6B). Login to comment
228 The relative expression levels of wt and mutant half-molecules were evaluated by densitometry and are indicated on the figure. B, effect of D792N and D1454N mutations on ATP-dependent LTC4 transport activity. Login to comment
229 Membrane vesicles (2 ␮g) containing wt and the D792N and D1454N mutant MRP1 half-molecules or control beta-gus were assayed for ATP-dependent LTC4 transport activity by incubation in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci) at 23°C for 2 min in the presence and absence of ATP (4 mM) as described under Materials and Methods. Results shown are means Ϯ S.D. of triplicate determinations in a single experiment. Login to comment
230 Similar results were obtained in three additional independent experiments. C, effect of D792N and D1454N mutations on photolabeling with 8-azido-[␣-32 P]ATP. Login to comment
242 The effect of the MRP1 Walker B D792N and D1454N mutations on ATP binding and ADP trapping was similar to that of the Walker A lysine mutations; the level of transport activity of the NBD1 aspartic acid-to-asparagine mutation was comparable with that of the Walker A lysine-to-methionine mutation (Gao et al., 2000). Login to comment
258 Densitometry indicated that the level of the D1454N mutant protein in the vesicle preparation used was approximately 2-fold higher than in control vesicles (A). Login to comment
260 B, effect of D792N and D1454N mutations on vanadate-dependent nucleotide trapping. Login to comment
265 C, effect of D792N and D1454N mutations on LTC4 photolabeling in the presence or absence of nucleotide. Login to comment
266 Membrane vesicles (50 ␮g of total protein) containing wt and the D792N and D1454N mutant MRP1 half-molecules were incubated in transport buffer at 23°C for 20 min in the absence or presence of ATP␥S (4 mM) or ATP (1 mM) plus vanadate (1 mM) before the addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment

[hide] Hydrogen-bond formation of the residue in H-loop o... Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18. Yang R, Chang XB
Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport.
Biochim Biophys Acta. 2007 Feb;1768(2):324-35. Epub 2006 Nov 18., [PMID:17187755]

Abstract [show]
MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
189 For example, ATP binding to wild-type MRP1 can transport the bound LTC4 from high to low affinity site whereas ATP binding to the K684R- or the D1454N-mutated MRP1 cannot [30]. 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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
241 Indeed, several mutations, such as K684E, K1333E, K684R, K1333R, D792N, D1454N, G771A and G1433A, significantly diminished ATP binding and Vi-dependent ADP trapping at NBD2 and lost the ability to shift the substrate binding from a high to low affinity site [61]. Login to comment

[hide] The hydroxyl group of S685 in Walker A motif and t... Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29. Yang R, Scavetta R, Chang XB
The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.
Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29., [PMID:18088596]

Abstract [show]
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 degrees C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the beta-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
254 In order to rule out the possibility that the double mutant D1454L/E1455L might rescue the misfolding caused by D14 54L mutation, we have made single mutants including D1454L, D1454N, S1334A, S1334T, S1334C, S1334H, S1334D and S1334N. Login to comment
261 It is also true for the corresponding mutations in Walker B motif of NBD2 in MRP1, such as D1454L (manuscript in preparation) and D1454N [46], have a significant effect on the ATP-dependent LTC4 transport. 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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
157 Indeed, several mutations, such as K684E, K1333E, K684R, K1333R, D792N, D1454N, G771A and G1433A, significantly diminished ATP binding and lost the ability to shift the bound substrate from high to low affinity site (99). Login to comment

[hide] ABCC multidrug transporters in childhood neuroblas... J Natl Cancer Inst. 2011 Aug 17;103(16):1236-51. Epub 2011 Jul 28. Henderson MJ, Haber M, Porro A, Munoz MA, Iraci N, Xue C, Murray J, Flemming CL, Smith J, Fletcher JI, Gherardi S, Kwek CK, Russell AJ, Valli E, London WB, Buxton AB, Ashton LJ, Sartorelli AC, Cohn SL, Schwab M, Marshall GM, Perini G, Norris MD
ABCC multidrug transporters in childhood neuroblastoma: clinical and biological effects independent of cytotoxic drug Efflux.
J Natl Cancer Inst. 2011 Aug 17;103(16):1236-51. Epub 2011 Jul 28., 2011-08-17 [PMID:21799180]

Abstract [show]
BACKGROUND: Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. METHODS: A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. RESULTS: Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis" expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). CONCLUSION: ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
66 For constitutive expression of ABCC1 in SH-SY5Y cells, transfection with pCMV14-3xFLAG-ABCC1, or the ATP-binding site mutants, pCMV14-3xFLAG-ABCC1-D1454N or ABCC1-DE1454LL, was followed by selection of stable clones with 500 µg/ml G418 (Geneticin; Invitrogen Australia Pty Ltd, Mulgrave, Australia), which were subsequently maintained with 150 µg/ml G418. Login to comment
67 The ABCC1-D1454N mutation was previously shown to abolish ATP-dependent ABCC1 transporter activity while not affecting correct localization of the transporter (19), and the ABCC1-DE1454LL mutant was predicted to have similar properties (Supplementary Figure 1, available online and http://www.uniprot.org/uniprot/O15439; accessed June, 2010). Login to comment
74 Generation of ATP-binding site mutants was carried out accordingtotheQuikChangeLightningSite-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) using forward primer 5'-ATC CTT GTG TTG AAT GAG GCC ACG GCA G-3' for ABCC1-D1454N (single mutant), forward primer 5'-ATC CTT GTG TTG CTT CTG GCC ACG GCA G-3' for ABCC1-DE1454/1455LL (double mutant) and forward primer 5'-AGG TGG GGA TCT TCG GCC GCA CTG G-3' for ABCC3-V1322F. Login to comment
151 ABCC1 transporter activity is necessary for the observed effects on cell motility, because forced expression of catalytically inactive ABCC1 (ABCC1-D1454N clone B4 or ABCC1-DE1454LL clone B7) had no impact on cell motility (mean % of wound open ± 95% CI: ABCC1 B4, 49.6 ± 4.5 vs empty vector, 59.9 ± 9.6, P = .08; ABCC1 B7, 55.6 ± 9.0 vs empty vector, 59.9 ± 9.6, P = .54, one-way ANOVA and two-sided t test, three independent Figure 2.Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. Login to comment
163 This effect was completely abrogated in cells overexpressing the mutant ABCC1 carrying a single amino acid change in the ABC domain (D1454N) or the catalytically inactive double mutant of ABCC1 (DE1454LL) (mean number of colonies ± 95% CI: ABCC1 B4, 74.8 ± 1.6 vs empty vector, 73.9 ± 4.3, P = .96; ABCC1 B7, 73.5 ± 5.9 vs empty vector, 73.9 ± 4.3, P = .88; one-way ANOVA and two-sided t test, four independent experiments; Figure 3, D). Login to comment
182 A) Western blot analysis of ABCC1 protein expression following stable transduction of SH-SY5Y cells with either empty vector, wild-type (wt) ABCC1 (clones D5, H7), ABCC1 D1454N single mutant (clone B4), or ABCC1 DD1454LL double mutant (clone B7) constructs. Login to comment
68 The ABCC1-D1454N mutation was previously shown to abolish ATP-dependent ABCC1 transporter activity while not affecting correct localization of the transporter (19), and the ABCC1-DE1454LL mutant was predicted to have similar properties (Supplementary Figure 1, available online and http://www.uniprot.org/uniprot/O15439; accessed June, 2010). Login to comment
75 Generation of ATP-binding site mutants was carried out accordingtotheQuikChangeLightningSite-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) using forward primer 5'-ATC CTT GTG TTG AAT GAG GCC ACG GCA G-3' for ABCC1-D1454N (single mutant), forward primer 5'-ATC CTT GTG TTG CTT CTG GCC ACG GCA G-3' for ABCC1-DE1454/1455LL (double mutant) and forward primer 5'-AGG TGG GGA TCT TCG GCC GCA CTG G-3' for ABCC3-V1322F. Login to comment
152 ABCC1 transporter activity is necessary for the observed effects on cell motility, because forced expression of catalytically inactive ABCC1 (ABCC1-D1454N clone B4 or ABCC1-DE1454LL clone B7) had no impact on cell motility (mean % of wound open ± 95% CI: ABCC1 B4, 49.6 ± 4.5 vs empty vector, 59.9 ± 9.6, P = .08; ABCC1 B7, 55.6 ± 9.0 vs empty vector, 59.9 ± 9.6, P = .54, one-way ANOVA and two-sided t test, three independent Figure 2.Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. Login to comment
164 This effect was completely abrogated in cells overexpressing the mutant ABCC1 carrying a single amino acid change in the ABC domain (D1454N) or the catalytically inactive double mutant of ABCC1 (DE1454LL) (mean number of colonies ± 95% CI: ABCC1 B4, 74.8 ± 1.6 vs empty vector, 73.9 ± 4.3, P = .96; ABCC1 B7, 73.5 ± 5.9 vs empty vector, 73.9 ± 4.3, P = .88; one-way ANOVA and two-sided t test, four independent experiments; Figure 3, D). Login to comment
183 A) Western blot analysis of ABCC1 protein expression following stable transduction of SH-SY5Y cells with either empty vector, wild-type (wt) ABCC1 (clones D5, H7), ABCC1 D1454N single mutant (clone B4), or ABCC1 DD1454LL double mutant (clone B7) constructs. Login to comment