ABCC2 p.Pro1158Ala
| Predicted by SNAP2: | A: N (61%), C: N (57%), D: D (66%), E: D (66%), F: D (53%), G: D (59%), H: D (59%), I: D (63%), K: D (66%), L: D (59%), M: D (59%), N: D (53%), Q: D (59%), R: D (63%), S: N (57%), T: D (53%), V: D (59%), W: D (71%), Y: D (59%), |
| 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, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Mutational analysis of a highly conserved proline ... Drug Metab Dispos. 2007 Aug;35(8):1372-9. Epub 2007 May 9. Letourneau IJ, Slot AJ, Deeley RG, Cole SP
Mutational analysis of a highly conserved proline residue in MRP1, MRP2, and MRP3 reveals a partially conserved function.
Drug Metab Dispos. 2007 Aug;35(8):1372-9. Epub 2007 May 9., [PMID:17494643]
Abstract [show]
The ATP-binding cassette multidrug resistance protein 1 MRP1 (ABCC1) mediates the cellular efflux of organic anions including conjugated metabolites, chemotherapeutic agents, and toxicants. We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C(4) (LTC(4)) transport but increased 17beta-estradiol 17beta-d-glucuronide (E(2)17betaG) and methotrexate (MTX) transport. Vanadate-induced trapping of [alpha-(32)P]8N(3)ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. To determine whether the functional importance of MRP1-Pro(1150) is conserved, the analogous Pro(1158) and Pro(1147) residues in the MRP2 and MRP3 transporters, respectively, were mutated to Ala. Expression levels of the three mutants were unaffected; however, the vesicular transport activity of at least one organic anion substrate was significantly altered. As observed for MRP1-Pro1150Ala, LTC(4) transport by MRP2-Pro1158Ala was decreased. However, E(2)17betaG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. In the case of MRP3-Pro1147Ala, LTC(4) transport was increased, whereas E(2)17betaG transport was unaffected. MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. In contrast, all three mutants showed a marked reduction in levels of vanadate-induced trapped [alpha-(32)P]8N(3)ADP. We conclude that MRP1-Pro(1150), MRP2-Pro(1158), and MRP3-Pro(1147) in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.
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No. Sentence Comment
5 As observed for MRP1-Pro1150Ala, LTC4 transport by MRP2-Pro1158Ala was decreased.
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ABCC2 p.Pro1158Ala 17494643:5:56
status: NEW122 As shown in Fig. 2, all three Pro to Ala mutants (MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala) were expressed at levels comparable to those of their corresponding wild-type proteins, confirming that the Pro residue at this position is not required for expression of the MRP proteins in the plasma membrane of mammalian cells.
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ABCC2 p.Pro1158Ala 17494643:122:72
status: NEW141 Protein expression levels of mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala and their corresponding wild-type proteins.
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ABCC2 p.Pro1158Ala 17494643:141:59
status: NEW148 Vesicular transport of 3 H-labeled organic anions by mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala.
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ABCC2 p.Pro1158Ala 17494643:148:83
status: NEW165 Mutations MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala Do Not Affect [␥-32 P]8N3ATP Binding but Decrease Vanadate-Induced Trapping of [␣-32 P]8N3ADP.
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ABCC2 p.Pro1158Ala 17494643:165:32
status: NEW175 Thus, 8N3ADP trapping by the MRP3 mutant was decreased as observed with the MRP1-Pro1150Ala and MRP2-Pro1158Ala mutants, although under different conditions.
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ABCC2 p.Pro1158Ala 17494643:175:101
status: NEW208 Thus, although 8N3ATP binding to MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala remained unchanged, the level of vanadate-induced 8N3ADP trapping was very low for all three mutant transporters.
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ABCC2 p.Pro1158Ala 17494643:208:55
status: NEW59 The template for generating the MRP2-Pro1158Ala mutant was created by subcloning a 2.2-kb ApaI/ClaI fragment into pGEM-7Zf(ϩ) (Promega, Madison, WI).
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ABCC2 p.Pro1158Ala 17494643:59:37
status: NEW62 Mutagenesis was performed according to the manufacturer`s instructions with the following sense primers (substituted nucleotides are underlined, and in both cases, the substitution created a BstUI restriction site, which was used to confirm the successful introduction of the nucleotide substitutions): MRP2-Pro1158Ala, 5Ј-C ACC AGG TCC GCG ATC TAC TCT C-3Ј (BstUI) and MRP3-Pro1147Ala, 5Ј-GTC AGC CGC TCC GCG ATC TAC TCC C-3Ј (BstUI).
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ABCC2 p.Pro1158Ala 17494643:62:308
status: NEW63 The MRP2-Pro1158Ala mutation was subcloned back into pcDNA3.1(-) MRP2 as a 1.75-kb Bsu36I/SfiI fragment and the MRP3-Pro1147Ala mutation was subcloned into pcDNA3.1(ϩ) MRP3 as a 1.24-kb AgeI/SacII fragment (Oleschuk et al., 2003).
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ABCC2 p.Pro1158Ala 17494643:63:9
status: NEW120 To determine whether this expression was also true for the corresponding MRP2-Pro1158Ala and MRP3-Pro1147Ala mutants, these mutations were created in pcDNA3.1(Ϯ)-based MRP2 and MRP3 expression vectors and then transfected into HEK293T cells.
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ABCC2 p.Pro1158Ala 17494643:120:78
status: NEW130 For MRP2-Pro1158Ala, LTC4 transport was also decreased by ϳ50% whereas it was significantly increased (1.8-fold) for MRP3-Pro1147Ala (Fig. 3A).
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ABCC2 p.Pro1158Ala 17494643:130:9
status: NEW137 In contrast to LTC4 uptake, E217betaG transport by MRP2 and MRP3 was unaffected by the Pro1158Ala and Pro1147Ala mutations, respectively (Fig. 3B).
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ABCC2 p.Pro1158Ala 17494643:137:87
status: NEW140 In contrast, MTX transport by the MRP2-Pro1158Ala mutant remained the same as that of wild-type MRP2 (Fig. 3C), whereas FIG. 2.
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ABCC2 p.Pro1158Ala 17494643:140:39
status: NEW160 To determine whether the reduced LTC4 transport by MRP2-Pro1158Ala was associated with reduced substrate binding, [3 H]LTC4 photolabeling of this mutant was compared with that of wild-type MRP2.
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ABCC2 p.Pro1158Ala 17494643:160:56
status: NEW163 Similarly, MRP2-Pro1158Ala was photolabeled by [3 H]LTC4 to the same extent as wild-type MRP2 despite the reduced LTC4 transport activity of the mutant (Fig. 4B).
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ABCC2 p.Pro1158Ala 17494643:163:16
status: NEW167 When MRP2-Pro1158Ala and MRP3-Pro1147Ala were photolabeled with [␥-32 P]8N3ATP, the intensity of the signals observed with the mutant and wild-type proteins was also comparable (Fig. 5), suggesting that ATP can bind to the Pro mutants as well as it does to their wild-type counterparts.
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ABCC2 p.Pro1158Ala 17494643:167:10
status: NEW170 When the MRP2-Pro1158Ala and MRP3-Pro1147Ala mutants were examined under the same conditions, a significant decrease (50%) in the level of vanadate-induced 8N3ADP trapping by MRP2-Pro1158Ala was also observed (Fig. 6B), whereas no trapping of nucleotide was detected with either wild-type MRP3 or its Pro1147Ala mutant (data not shown).
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ABCC2 p.Pro1158Ala 17494643:170:14
status: NEWX
ABCC2 p.Pro1158Ala 17494643:170:180
status: NEW[hide] ABCC2/Abcc2: a multispecific transporter with domi... Drug Metab Rev. 2010 Aug;42(3):402-36. Jemnitz K, Heredi-Szabo K, Janossy J, Ioja E, Vereczkey L, Krajcsi P
ABCC2/Abcc2: a multispecific transporter with dominant excretory functions.
Drug Metab Rev. 2010 Aug;42(3):402-36., [PMID:20082599]
Abstract [show]
ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics.
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No. Sentence Comment
97 Mutant Predicted location Substrate Activity changes Reference Human MRP2 Δ1-188 TMD0 LTC4 ↓ Fernandez et al., 2002 K316A JC, TM6 GMF ↔ Ryu et al., 2000 K324A TM6 GMF ↓ Ryu et al., 2000 K329A TM6 GMF ↔ Ryu et al., 2000 R412G DJ IC MTX ↓ Hulot et al., 2005 W417I IC, TM7-TM8 E2-17βG ↓ Hirouchi et al., 2004 LTC4 ↓ DNP-SG ↓ H439A TM8 GMF ↔ Ryu et al., 2000 K483A IC, JM, TM9 GMF ↓ Ryu et al., 2000 K590A JC, TM11 GMF ↔ Ryu et al., 2000 S789F NBD1 E2-17βG ↓ Hirouchi et al., 2004 LTC4 ↓ DNP-SG ↓↓ R1023A EC, JM, TM13 GMF ↔ Ryu et al., 2000 H1042A TM13 GMF ↔ Ryu et al., 2000 R1100A JC, TM14 GMF ↔ Ryu et al., 2000 P1158A IC, JM, TM15 LTC4 ↓↓ Letourneau et al., 2007 E2-17βG ↔ MTX ↔ Table 1. continued on next page Mutant Predicted location Substrate Activity changes Reference I1173F DJ IC, TM15-16 LTC4 No act Keitel et al., 2003 E2-17βG No act R1210A EC, JC, TM16 GMF ↓↓ Ryu et al., 2000 R1230A TM16 GMF ↔ R1257A JC, TM17 GMF ↓↓ W1254A JC, TM17 E2-17βG ↓↓ Ito et al., 2001a W1254C ↓↓ W1254F ↔ W1254Y ↔ W1254A JC, TM17 LTC4 ↓↓ Ito et al., 2001b W1254C ↓↓↓ W1254F ↓↓ W1254Y ↓↓ W1254A JC, TM17 MTX ↓↓ Ito et al., 2001a W1254C ↓↓ W1254F ↓↓ W1254Y ↓↓↓ A1450T NBD2 E2-17βG ↓↓ Hirouchi et al., 2004 LTC4 ↓↓ DNP-SG ↓↓ Rat Mrp2 K308M IC, JM, TM6 TLC-S ↔ Ito et al., 2001b DNP-G ↑ LTC4 ↓ E3040G ↔ K320M TM6 TLC-S ↑ DNP-G ↑ LTC4 ↓ E3040G ↑ K325M TM6 TLC-S ↓* DNP-G ↓↓↓* LTC4 ↓↓↓* E3040G ↓ D329N TM6 TLC-S ↔ DNP-G ↓ LTC4 ↓↓↓* E3040G ↓ R586L TM11 TLC-S ↓ DNP-G ↓↓* LTC4 ↓↓* E3040G ↔ R1019M IC, JM, TM13 TLC-S ↔ DNP-G ↑* LTC4 ↔ E3040G ↔ R1096L TM14 TLC-S ↑ DNP-G ↑ LTC4 ↔ E3040G ↔ EC, extracellular; IC, intracellular; JC, near the cytosol in the membrane; JM, juxtamembrane; TLC-S, tauro-litocholate-sulfate; GMF, glutathione- methyl-fluorescein; ↑, activity over control>1.2; ↔, 1.2>activity over control>0.8; ↓, 0.8>activity over control>0.5; ↓↓, 0.5>activity over control>0.1; ↓↓↓, 0.1>activity over control.
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ABCC2 p.Pro1158Ala 20082599:97:755
status: NEW