ABCC1 p.Arg1197Lys
| Predicted by SNAP2: | A: D (85%), C: D (75%), D: D (95%), E: D (91%), F: D (91%), G: D (85%), H: D (80%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (85%), P: D (95%), Q: D (91%), S: D (80%), T: D (85%), V: D (91%), W: D (95%), Y: D (91%), |
| 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, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]
Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.
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No. Sentence Comment
118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details).
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ABCC1 p.Arg1197Lys 15208328:118:555
status: NEW146 To determine whether the charge or size of the Arg1197 and Arg1249 side chains were important for MRP1 transport function, the like-charge substituted mutants R1197K and R1249K were created.
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ABCC1 p.Arg1197Lys 15208328:146:159
status: NEW148 Nevertheless, ATP-dependent E217betaG uptake by the R1197K and R1249K mutants was 20 and Ͻ10% of wild-type MRP1, respectively (Fig. 5A).
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ABCC1 p.Arg1197Lys 15208328:148:52
status: NEW149 Similarly, uptake levels of LTC4 (Fig. 5B), E1SO4 (Fig. 5C), and MTX (Fig. 5D) by the R1197K and R1249K mutants were reduced to Յ10% of wild-type MRP1 uptake levels.
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ABCC1 p.Arg1197Lys 15208328:149:86
status: NEW151 Effect of Glu1204 , Arg1197 , and Arg1249 Mutations on Photolabeling with [3 H]LTC4 and 8-Azido-[␣-32 P]ATP-In the next series of experiments, those same-charge or neutrally substituted mutants that showed substantially reduced transport activities (R1197K, E1204L, and R1249K) were further examined to determine whether their loss of transport activity was accompanied by a decrease in substrate binding.
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ABCC1 p.Arg1197Lys 15208328:151:257
status: NEW152 As shown in Fig. 6A, [3 H]LTC4 photolabeling of the R1197K and R1249K mutants was completely abrogated, indicating that the reduced LTC4 transport activity of these mutants was associated with decreased binding of this substrate.
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ABCC1 p.Arg1197Lys 15208328:152:52
status: NEW154 To determine whether the mutations of Arg1197 , Glu1204 , and Arg1249 that altered the transport properties of MRP1 also affected the interaction of the transporter with nucleotide, the ability of the R1197K, E1204L, and R1249K mutants to be photolabeled with 8-azido-[␣-32 P]ATP, both at 4 °C to minimize hydrolysis and at 37 °C in the presence of sodium vanadate to trap azido-ADP after hydrolysis, was examined (31, 32).
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ABCC1 p.Arg1197Lys 15208328:154:201
status: NEW155 As shown in Fig. 6B, 8-azido-[␣-32 P]ATP labeling of the R1197K and R1249K mutants was comparable with wild-type MRP1.
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ABCC1 p.Arg1197Lys 15208328:155:64
status: NEW157 Furthermore, orthovanadate-induced trapping of 8-azido-[␣- 32 P]ADP of the R1197K and R1249K mutants was also comparable with wild-type MRP1 (Fig. 6C).
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ABCC1 p.Arg1197Lys 15208328:157:82
status: NEW177 Levels of 3 H-labeled organic anion uptake by membrane vesicles were prepared from cells expressing wild-type MRP1 (black bars), mutants R1197E, R1197K, R1249D, and R1249K (gray bars), and empty pcDNA3.1 vector control vesicles (open bars).
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ABCC1 p.Arg1197Lys 15208328:177:145
status: NEW192 A, membrane vesicle proteins (50 g) prepared from cells expressing wild-type (WT-MRP1) and transport-compromised mutant MRP1 proteins (R1197K, E1204L, and R1249K) were incubated with [3 H]LTC4 (200 nM; 250 nCi) followed by UV cross-linking, SDS-PAGE, and fluorography.
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ABCC1 p.Arg1197Lys 15208328:192:143
status: NEW232 The lack of LTC4 labeling of the R1197K and R1249K mutants indicates that their binding site for LTC4 (and likely other organic anions) has been disrupted.
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ABCC1 p.Arg1197Lys 15208328:232:33
status: NEW234 The bulkier, less ionizable Lys side chains in the R1197K and R1249K mutants presumably either cannot form or are prevented from forming the interhelical and/or intrahelical interactions that are established by Arg in wild-type MRP1 for proper folding into a functional transporter.
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ABCC1 p.Arg1197Lys 15208328:234:51
status: NEW[hide] Mutational analysis of polar amino acid residues w... Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13. Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SP, Deeley RG
Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity.
Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13., [PMID:16415113]
Abstract [show]
Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.
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No. Sentence Comment
164 Conversely, replacement of Glu1204 with Leu or Arg1197 with Glu or Lys affected either substrate specificity or overall transport activity of MRP1 (Situ et al., 2004).
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ABCC1 p.Arg1197Lys 16415113:164:47
status: NEW[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
116 The R1197K mutation within or near TM16 almost completely abolished LTC4 binding and ATP-dependent LTC4 transport [83].
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ABCC1 p.Arg1197Lys 17295059:116:4
status: NEW[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.
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ABCC1 p.Arg1197Lys 19949927:104:296
status: NEW