ABCMdb

ABCC1 p.Asn590Ala

Predicted by SNAP2:	A: D (71%), C: D (71%), D: D (71%), E: D (80%), F: D (85%), G: D (66%), H: D (66%), I: D (80%), K: D (85%), L: D (85%), M: D (71%), P: D (85%), Q: D (63%), R: D (85%), S: D (59%), T: D (63%), V: D (85%), W: D (91%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Transmembrane helix 11 of multidrug resistance pro... Biochemistry. 2004 Jul 27;43(29):9413-25. Zhang DW, Nunoya K, Vasa M, Gu HM, Theis A, Cole SP, Deeley RG
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1.
Biochemistry. 2004 Jul 27;43(29):9413-25., 2004-07-27 [PMID:15260484]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

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No. Sentence Comment
10 Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC4) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Login to comment
47 Ser604 was converted to Ala and Thr, and Asn590 was mutated to Ala, Gln, and Asp. Login to comment
60 Mutations S585A, N590A, N590Q, N590D, N597A, and S604A were generated using the QuikchangeSite-Directed Mutagenesis kit (STRATAGENE, La Jolla, CA). Login to comment
64 They are as follows: S585A (5'-C CAG ACA GCC TTC GTG GCT TTG GCC TTG-3), N590A (5'- CT TTG GCC TTG TTC GCC ATC CTC CGG TTT CCC-3'), N590Q (5'-CT TTG GCC TTG TTC CAG ATC CTC FIGURE 1: Topology of human MRP1. Login to comment
94 To generate the MRP1N590A -pFASTBAC dual vector, pCEBV7-MRP1 containing mutation N590A was digested with BamHI and SphI, and an approximately 1.8 kb fragment comprised of nucleotides 840-2699 of MRP1N590A was isolated. Login to comment
152 The only mutation that affected LTC4 transport was replacement of Asn590 by Ala, which decreased the ability of MRP1 to transport LTC4 by approximately 50-60% (Figure 3A-D). Login to comment
156 On the other hand, replacement of Asn590 with Ala (N590A) substantially decreased the levels of E217 G transport. Login to comment
157 Thus, mutation S604A altered only the ability of MRP1 to transport the glucuronidated estrogen, whereas mutation N590A influenced the transport of both LTC4 and E217 G. Login to comment
168 To examine the influence of the N590A and S604A mutations more quantitatively, we compared their effect on the kinetic parameters of transport of both substrates (Figure 5). Login to comment
170 In contrast, the N590A mutation decreased the Vmax and increased the apparent Km value for LTC4 transport (56 pmol/mg/min and 277 nM for mutation N590A) (Figure 5B and Table 1). Login to comment
171 Thus, mutation N590A decreased the Vmax/Km ratio for LTC4 approximately 3-fold. Login to comment
173 In contrast, substitution of Asn590 with Ala increased the Km value by approximately 65% (2.8 µM) and decreased the Vmax value by approximately 2-fold (93 pmol/mg/min) (Figure 5C and Table 1). Login to comment
174 Thus, as observed with LTC4 as a substrate, mutation N590A decreased the Vmax/Km ratio for E217 G approximately 3-fold, whereas the S604A mutation increased the Vmax/Km ratio for this substrate approximately 3-fold. Login to comment
175 These changes in transport efficiency were mediated predominantly by a decrease in apparent affinity for both LTC4 and E217 G as a result of the S604A mutation, while the N590A mutation increased the apparent Km and decreased the Vmax for E217 G. Login to comment
177 Since transport studies indicated that mutation N590A increased the apparent Km for LTC4, we directly examined the ability of the mutant protein to bind and be photolabeled by [3 H]LTC4. Login to comment
178 The N590A mutation decreased photolabeling, consistent with the results obtained with kinetic analysis of LTC4 uptake (Figure 6B). Login to comment
180 Although photolabeling of the N590A mutant protein was decreased relative to wild type in the absence of ATP, a further decrease was observed in the presence of nucleotide, indicating that the protein retains the ability to shift from a higher to lower affinity state. Login to comment
181 To investigate the effect of mutation N590A on photolabeling of LTC4 more precisely, we took advantage of a pFASTBAC dual vector, in which the NH2-proximal MRP1 fragment (amino acids 1-932) was modified to contain mutation N590A and coexpressed with a wild-type COOH-proximal fragment (amino acids 932-1531). Login to comment
196 As observed with results obtained from analyses of membrane vesicles prepared from HEK293 cells stably transfected with full-length mutant MRP1N590A , replacement of Asn590 by Ala decreased the transport activity by approximately 50%. Login to comment
198 As shown in Figure 6E, the N590A mutation resulted in a significant reduction of the labeling of LTC4 at both the NH2-and the COOH-proximal halves of MRP1. Login to comment
215 The studies described previously showed that the N590A mutation modulated the ability of MRP1 to confer drug resistance and to transport conjugated organic anions. Login to comment
220 As observed with the effects of the mutations on LTC4 transport, only replacement of Asn590 with Ala significantly decreased the ability of MRP1 to transport GSH (Figure 7). Login to comment
222 Effect of Mutation N590A on Photolabeling of MRP1 with 8-Azido-[R32 P]ATP. Login to comment
223 On the basis of all substrates tested, mutation N590A affected the overall activity of MRP1. Login to comment
225 To investigate whether the N590A mutation influenced the binding and/or hydrolysis of ATP at NBD1 and/or NBD2 FIGURE 4: ATP-dependent [3H]LTC4 and [3H]E217 G uptake by membrane vesicles prepared from HEK293 cells transfected with wild-type or mutant MRP1. Login to comment
240 As shown in Figure 8B, substitution of Asn590 with Ala dramatically decreased the binding of the ATP analogue at NBD1. Login to comment
245 However, as shown in Figure 8C, no significant difference could be detected between wild-type and N590A mutant proteins with respect to the ability of the LTC4 to enhance ADP trapping at NBD2 (compare lanes 5 and 6 and lanes 2 and 3 in Figure 8C) (46). Login to comment
249 In contrast, rather than altering substrate specificity, replacement of Asn590 with Ala uniformly decreased the overall activity of MRP1, both with respect to transport of organic anion conjugates and resistance to three different classes of drugs. Login to comment
250 Thus, the consequences of the N590A mutation are similar to those of nonconservative mutations of other amino acids (Arg593 , Phe594 , and Pro595 ) predicted to be near the middle of TM11 (35, 48, 49). Login to comment
276 The conversion of Asn590 to Ala substantially decreased the ability to confer drug resistance and to transport E217 G, LTC4, and GSH. Login to comment
289 Given the pleiotropic effect of substitution of Asn590 with Ala and the lack of an effect of replacement with Asp or Gln, it appears likely that the hydrogen-bonding capacity of the side chain of the residue at position 590 may play a role in defining the conformation of the protein in the vicinity of the substrate binding pocket, possibly by interacting with residues in another helix. On the basis of our recently developed model of the tertiary structure of MSD2 and MSD3 of MRP1, such an interaction appears most likely to involve residues in TM6 and TM10 (48). Login to comment
293 The uniform effect of the N590A mutation on the relative efficiency of transport of several structurally diverse conjugated anions, as well as resistance to three different classes of drugs, suggested that it may have altered a common function, such as the coupling of transport to ATP hydrolysis or nucleotide binding and hydrolysis itself. Login to comment
313 The effect of the N590A mutation is less severe, and the mutant protein retains 30-40% of the transport activity of wild-type MRP1. Login to comment
315 Thus, it appears likely that the reduction in transport in the N590A mutation is attributable to a decrease in the binding of ATP by NBD1 and a consequential reduction in ATP binding and hydrolysis at NBD2. Login to comment
319 Since there are many functional similarities between the cooperative interactions of the NBDs of MRP1 and CFTR, it is possible that the CFTR R347D mutation may be affecting the same step in the catalytic cycle as the N590A mutation. Login to comment
324 Thus, changes in the position or orientation of TM11 caused by mutations, such as N590A, may be transmitted through the ICD causing a conformational or positional change in NBD1 that alters cooperativity between the two NBDs and results in diminished binding of ATP. Login to comment

[hide] Substrate recognition and transport by multidrug r... FEBS Lett. 2006 Feb 13;580(4):1103-11. Epub 2005 Dec 21. Deeley RG, Cole SP
Substrate recognition and transport by multidrug resistance protein 1 (ABCC1).
FEBS Lett. 2006 Feb 13;580(4):1103-11. Epub 2005 Dec 21., 2006-02-13 [PMID:16387301]

Abstract [show]
Multidrug resistance protein (MRP) 1 belongs to the 'C' branch of the ABC transporter superfamily. MRP1 is a high-affinity transporter of the cysteinyl leukotriene C(4) and is responsible for the systemic release of this cytokine in response to an inflammatory stimulus. However, the substrate specificity of MRP1 is extremely broad and includes many organic anion conjugates of structurally unrelated endo- and xenobiotics. In addition, MRP1 transports unmodified hydrophobic compounds, such as natural product type chemotherapeutic agents and mutagens, such as aflatoxin B(1). Transport of several of these compounds has been shown to be dependent on the presence of reduced glutathione (GSH). More recently, GSH has also been shown to stimulate the transport of some conjugated compounds, including sulfates and glucuronides. Here, we summarize current knowledge of the substrate specificity and modes of transport of MRP1 and discuss how the protein may recognize its structurally diverse substrates.

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No. Sentence Comment
146 However, alanine substitution of Asn590 affects transport of a variety of substrates, apparently by reducing the binding of ATP by NBD1 [76], although other amino acid substitutions have no effect. Login to comment

[hide] Transmembrane transport of endo- and xenobiotics b... Physiol Rev. 2006 Jul;86(3):849-99. Deeley RG, Westlake C, Cole SP
Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins.
Physiol Rev. 2006 Jul;86(3):849-99., [PMID:16816140]

Abstract [show]
Multidrug Resistance Proteins (MRPs), together with the cystic fibrosis conductance regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9) comprise the 13 members of the human "C" branch of the ATP binding cassette (ABC) superfamily. All C branch proteins share conserved structural features in their nucleotide binding domains (NBDs) that distinguish them from other ABC proteins. The MRPs can be further divided into two subfamilies "long" (MRP1, -2, -3, -6, and -7) and "short" (MRP4, -5, -8, -9, and -10). The short MRPs have a typical ABC transporter structure with two polytropic membrane spanning domains (MSDs) and two NBDs, while the long MRPs have an additional NH2-terminal MSD. In vitro, the MRPs can collectively confer resistance to natural product drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and, under certain circumstances, alkylating agents. The MRPs are also primary active transporters of other structurally diverse compounds, including glutathione, glucuronide, and sulfate conjugates of a large number of xeno- and endobiotics. In vivo, several MRPs are major contributors to the distribution and elimination of a wide range of both anticancer and non-anticancer drugs and metabolites. In this review, we describe what is known of the structure of the MRPs and the mechanisms by which they recognize and transport their diverse substrates. We also summarize knowledge of their possible physiological functions and evidence that they may be involved in the clinical drug resistance of various forms of cancer.

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No. Sentence Comment
849 Similarly, only Ala substitution of Asn590 (a "cavity"-creating substitution) adversely affected MRP1 activity, while replacing this residue with Asp or Gln had no effect, suggesting that the polar side chain of Asn590 may be involved in interhelical interactions that influence the conformation of the protein in the vicinity of the binding pocket (570). Login to comment
848 Similarly, only Ala substitution of Asn590 (a "cavity"-creating substitution) adversely affected MRP1 activity, while replacing this residue with Asp or Gln had no effect, suggesting that the polar side chain of Asn590 may be involved in interhelical interactions that influence the conformation of the protein in the vicinity of the binding pocket (570). Login to comment
850 Similarly, only Ala substitution of Asn590 (a "cavity"-creating substitution) adversely affected MRP1 activity, while replacing this residue with Asp or Gln had no effect, suggesting that the polar side chain of Asn590 may be involved in interhelical interactions that influence the conformation of the protein in the vicinity of the binding pocket (570). 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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No. Sentence Comment
112 Many mutations in TM11, such as N590A, F594A, N597A, S604A and S605A, also modulate the drug resistance profile of MRP1 [79, 80]. 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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No. Sentence Comment
104 Mutations of C43S in TM1 (112); P343A, K332L and K332D in TM6 (113, 114); W445A and P448A in TM8 (113, 115); T550A, T556A and P557A in TM10 (113, 116); N590A, F594A, P595A, N597A, S604A and S605A in TM11 (113, 117, 118); E1089Q, E1089A, E1089L, E1089N, K1092, S1097 and N1100 in TM14 (119, 120); R1197K in TM16 (121); Y1236F, T1241A, T1242A, T1242C, T1242S, T1242L, Y1243F, N1245A, W1246C, W1246A, W1246F, W1246Y or R1249K in TM17 (121-124) significantly affect MRP1 function. Login to comment