ABCC1 p.Pro1150Ala
| Predicted by SNAP2: | A: N (57%), C: N (57%), D: D (66%), E: D (66%), F: D (59%), G: D (63%), H: D (63%), I: D (63%), K: D (66%), L: D (59%), M: D (59%), N: D (59%), Q: D (59%), R: D (63%), S: N (57%), T: D (59%), V: D (63%), 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] Identification of proline residues in the core cyt... J Biol Chem. 2004 Mar 26;279(13):12325-36. Epub 2004 Jan 13. Koike K, Conseil G, Leslie EM, Deeley RG, Cole SP
Identification of proline residues in the core cytoplasmic and transmembrane regions of multidrug resistance protein 1 (MRP1/ABCC1) important for transport function, substrate specificity, and nucleotide interactions.
J Biol Chem. 2004 Mar 26;279(13):12325-36. Epub 2004 Jan 13., 2004-03-26 [PMID:14722114]
Abstract [show]
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro(343), TM8-Pro(448), TM10-Pro(557), and TM11-Pro(595)) and MSD3 (TM14-Pro(1088)) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro(1150) in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17beta-estradiol-17-beta-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.
Comments [show]
None has been submitted yet.
No. Sentence Comment
7 Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed.
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ABCC1 p.Pro1150Ala 14722114:7:93
status: NEW49 The altered transport activity of P1150A was associated with significant changes in the ATP dependence of E217betaG but not LTC4 transport, suggesting that CL7 may play a differential role in coupling the activity of the MRP1 NBDs to the translocation of different substrates across the membrane.
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ABCC1 p.Pro1150Ala 14722114:49:34
status: NEW58 Position MRP1 MRP3 MRP2 MRP6 SUR1 SUR2 MRP7 MRP4 MRP5 CFTR MSD2 323 Pro Ser Met Ser Gly Gly Arg Lys Thr Trp 343 Pro Pro Pro Pro Pro Asn Pro Pro Pro Pro 359 Pro Pro Tyr Pro Phe Phe Pro Ala Asn Glu 448 Pro Pro Val Leu Pro Pro Pro Pro Pro Pro 464 Pro Pro Pro Pro Val Ser Val Ile Pro Ala 478 Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu 557 Pro Pro Pro Thr Pro Pro Pro Ser Val Gly 595 Pro Pro Pro Ala Pro Pro Pro Thr Ala Ala 600 Pro Pro Pro Pro Ser Phe Pro Phe Pro Phe MSD3 1003 Pro Ala Ser Pro Ala Lys Gln Gly Gln 1060 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1068 Pro Pro Pro Pro Pro Pro Pro Pro Pro Lys 1088 Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro 1113 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1120 Pro Leu Ile Leu Leu Leu Pro Val Gly Val 1121 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1150 Pro Pro Pro Ser Pro Pro Pro Pro Pro Pro 1191 Pro Pro Ser Pro Phe Phe Ala Leu Leu Leu AGC AGC-3Ј; and MSD3 Pro mutants B P1003A, 5Ј-C TGG ACT GAT GAC GCC ATC GTC AAC GGG-3Ј; P1060A, 5Ј-C CTG CGG TCA GCC ATG AGC TTC-3Ј; P1068A, 5Ј-C TTT GAG CGG ACC GCG AGT GGG AAC C-3Ј; P1088A, 5Ј-C TCC ATG ATC GCG GAG GTC ATC-3Ј; P1113A, 5Ј-CTG CTG GCC ACG GCC ATC GCC GCC-3Ј; P1120A, 5Ј-GCC ATC ATC ATC GCG CCC CTT GG-3Ј; P1121A, 5Ј-C ATC ATC ATC CCG GCG CTT GGC CTC ATC-3Ј; P1150A, 5Ј-G GTC AGC CGC TCC GCG GTC TAT TCC C-3Ј; P1191A, 5Ј-C CAG AAG GCC TAT TAC GCT AGC ATC GTG GCC AAC-3Ј; P1120A/P1121A, 5Ј-C GCC ATC ATC ATC GCA GCG CTT GGC CTC ATC TAC TTC-3Ј.
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ABCC1 p.Pro1150Ala 14722114:58:1332
status: NEW139 The locations of the MSD3 Pro residues mutated in this study are highlighted, and the approximate boundaries of the TM helices are indicated by dashed lines. B, shown is a representative immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (P1003A, P1060A, P1068A, P1088A, P1113A, P1120A, P1121A, P1150A, and P1191A) MRP1 cDNAs. MRP1 proteins were detected with mAb QCRL-1, and relative levels of expression estimated by densitometry are indicated; equal protein loading was confirmed by Amido Black staining of the polyvinylidene difluoride membrane and is shown below the blot.
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ABCC1 p.Pro1150Ala 14722114:139:381
status: NEW147 As shown in Fig. 4A, [3 H]LTC4 uptake levels of the P1003A, P1068A, P1088A, and P1150A mutants were ϳ40-50% less than those of wild-type MRP1, whereas LTC4 uptake by the other five MSD3 Pro mutants was similar to wild-type MRP1.
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ABCC1 p.Pro1150Ala 14722114:147:80
status: NEW149 In marked contrast, uptake of [3 H]E217betaG by the P1150A mutant was 2.2-fold higher than wild-type MRP1 (Fig. 4B).
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ABCC1 p.Pro1150Ala 14722114:149:52
status: NEW150 [3 H]MTX uptake by the P1150A mutant was also dramatically increased (ϳ6-fold) whereas uptake of this antifolate by the remaining eight MSD3 Pro mutants was either moderately reduced (by 50-60% for P1068A, P1088A, and P1120A) or comparable with wild-type MRP1 (P1003A, P1060A, P1113A, P1121A, and P1191A) (Fig. 4C).
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ABCC1 p.Pro1150Ala 14722114:150:23
status: NEW151 To determine whether Ala substitution of Pro1150 also affected the transport of other folic acid derivatives, [3 H]leucovorin uptake by the P1150A mutant was assessed.
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ABCC1 p.Pro1150Ala 14722114:151:21
status: NEWX
ABCC1 p.Pro1150Ala 14722114:151:140
status: NEW152 As shown in Fig. 4D, levels of [3 H]leucovorin uptake by wild-type and P1150A mutant MRP1 were similar, indicating that the enhanced MTX transport activity caused by the Pro1150 substitution is quite substrate-specific.
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ABCC1 p.Pro1150Ala 14722114:152:71
status: NEW154 Relative to wild-type MRP1, apigenin-stimulated GSH uptake by the P1068A, P1088A, and P1150A mutants was significantly reduced (by 50-70%), whereas uptake by the other six MSD3 Pro FIG. 4.
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ABCC1 p.Pro1150Ala 14722114:154:86
status: NEW159 TABLE III Summary of effects of MSD3 Pro substitutions on MRP1 vesicular transport activities Substrate % wild-type MRP1 activitya ECL6 P1003A CL6 TM14 P1088A ECL7 P1113A TM15 CL7 P1060A P1068A P1120A P1121A P1150A P1191A LTC4 60 90 60 50 100 100 110 50 90 E217betaG 65 50 55 45 90 60 70 220 90 MTX 80 80 40 45 100 50 85 620 100 GSH 80 95 50 30 120 140 80 35 100 E13SO4 75 75 90 50 120 90 80 65 60 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in 2-3 independent experiments (for details, see legend to Fig. 4 and text).
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ABCC1 p.Pro1150Ala 14722114:159:208
status: NEW162 GSH-stimulated E13SO4 uptake by the P1003A, P1060A, P1088A, P1150A, and P1191A mutants was moderately reduced (by 25-50%), whereas uptake of this sulfated estrogen by the other four MSD3 Pro mutants (P1068A, P1113A, P1120A, and P1121A) was comparable with wild-type MRP1.
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ABCC1 p.Pro1150Ala 14722114:162:60
status: NEW185 The horizontal white scale bar in the image represents 20 m. Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Uptake by MRP1 Pro Mutants-MSD2 TM mutants P343A, P448A, P478A, P557A, and P595A, MSD3 CL7 mutant P1150A, and TM14 mutant P1088A whose [3 H]LTC4 or [3 H]E217betaG transport properties were substantially altered relative to wild-type MRP1 were further characterized by kinetic analyses (Table IV).
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ABCC1 p.Pro1150Ala 14722114:185:219
status: NEW193 The apparent Km(LTC4) values for the MSD3 mutants P1088A and P1150A were also somewhat decreased (55 and 40 nM, respectively) compared with wild-type MRP1 (72 nM), as were their Vmax(LTC4) values (by ϳ50%), yielding overall LTC4 transport efficiencies (Vmax/Km) for these two mutants of 4.15 and 4.55, respectively, compared with a value of 5.86 for wild-type MRP1.
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ABCC1 p.Pro1150Ala 14722114:193:61
status: NEW194 The apparent Km(E217betaG) for the MSD3 P1150A mutant, which showed significantly enhanced E217betaG transport, was decreased 4-fold (0.24 versus 1.02 M for wild-type MRP1).
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ABCC1 p.Pro1150Ala 14722114:194:40
status: NEW196 On the other hand, the Vmax(E217betaG) values of P1150A and P1088A (160 and 165 pmol mg-1 min-1 , respectively) were comparable with wild-type MRP1 (176 pmol mg-1 min-1 ).
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ABCC1 p.Pro1150Ala 14722114:196:49
status: NEW197 Thus, the overall E217betaG transport efficiency of P1150A was enhanced ϳ4-fold whereas that of P1088A was reduced by 35%, largely because of changes in the apparent uptake affinity of the mutant proteins for this conjugated estrogen.
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ABCC1 p.Pro1150Ala 14722114:197:52
status: NEW198 Photolabeling of Wild-type and Pro Mutant MRP1 Proteins with [3 H]LTC4-To investigate further whether the reduced [3 H]LTC4 transport activity of the TM mutants P343A, P448A, P478A, P557A, P595A, and P1088A and the CL7 mutant P1150A was associated with a decrease in substrate binding, photolabeling experiments were carried out with this intrinsically photoactivable arachidonic acid derivative.
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ABCC1 p.Pro1150Ala 14722114:198:226
status: NEW201 ATP Dependence of LTC4 and E217betaG Transport by MRP1-P1150A-To explore further the mechanism of enhanced E217betaG transport by the P1150A mutant, the ATP dependence of E217betaG uptake by membrane vesicles expressing this mutant MRP1 was investigated by measuring initial rates of up- FIG. 7.
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ABCC1 p.Pro1150Ala 14722114:201:55
status: NEWX
ABCC1 p.Pro1150Ala 14722114:201:134
status: NEW204 Radiolabeled vesicles enriched for wild-type MRP1 (WT-MRP1) and MRP1 mutants P343A, P448A, P1088A, P1150A, and empty vector control (pcDNA3.1(-)) are shown in the upper panel, and radiolabeled vesicles enriched for WT-MRP1 and mutants P478A, P557A, P595A, P1150A, and pcDNA3.1(-) are shown in the lower panel.
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ABCC1 p.Pro1150Ala 14722114:204:99
status: NEWX
ABCC1 p.Pro1150Ala 14722114:204:256
status: NEW205 TABLE IV Kinetic parameters of vesicular LTC4 and E217beta G uptake by selected Pro mutants of MRP1 Km Vmax Vmax/ Km Mutant:WT nM pmol mg-1 min-1 ϫ10-3 Vmax/Km LTC4 WT-MRP1 72 422 5.86 1.0 P343A 50 203 4.06 0.7 P448A 39 155 3.97 0.7 P1088A 55 229 4.15 0.7 P1150A 40 182 4.55 0.8 WT-MRP1 115 674 5.85 1.0 P478A 49 160 3.24 0.6 P557A 63 191 3.02 0.5 P595A 485 239 0.49 0.1 E217betaG WT-MRP1 1017 176 0.17 1.0 P1088A 1390 165 0.12 0.7 P1150A 243 160 0.66 3.9 take of this conjugated organic anion at different ATP concentrations (Fig. 8A).
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ABCC1 p.Pro1150Ala 14722114:205:262
status: NEWX
ABCC1 p.Pro1150Ala 14722114:205:438
status: NEW206 The ATP dependence of LTC4 transport by P1150A was also measured for comparison (Fig. 8B).
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ABCC1 p.Pro1150Ala 14722114:206:40
status: NEW207 In the case of E217betaG transport (Fig. 8A), the Km(ATP) and Vmax values were 108 M and 295 pmol mg-1 min-1 and 483 M and 142 pmol mg-1 min-1 for P1150A and wild-type MRP1, respectively.
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ABCC1 p.Pro1150Ala 14722114:207:163
status: NEW208 In the case of LTC4 transport (Fig. 8B), the Km(ATP) and Vmax values were 97 M and 90 pmol mg-1 min-1 and 109 M and 170 pmol mg-1 min-1 for P1150A and wild-type MRP1, respectively.
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ABCC1 p.Pro1150Ala 14722114:208:156
status: NEW209 Thus the MRP1-P1150A mutant showed a significantly lower (nearly 5-fold) Km (higher apparent affinity) for ATP than wild-type MRP1 when supporting transport of E217betaG.
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ABCC1 p.Pro1150Ala 14722114:209:14
status: NEW210 In contrast, when supporting LTC4 transport, the Km(ATP) of P1150A and wild-type MRP1 were similar.
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ABCC1 p.Pro1150Ala 14722114:210:60
status: NEW211 8-Azido-[␣-32 P]ATP Binding and Orthovanadate-induced Trapping of 8-Azido-[␣-32 P]ADP by MRP1-P1150A-To investigate further the ATP binding and hydrolysis properties of the MRP1-P1150A mutant, we used a 32 P-labeled photoactivable nucleotide analog to evaluate its relative ability to bind ATP.
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ABCC1 p.Pro1150Ala 14722114:211:108
status: NEWX
ABCC1 p.Pro1150Ala 14722114:211:192
status: NEW212 As shown in Fig. 9A, at 4 °C 8-azido-[␣-32 P]ATP photolabeled a 190-kDa protein corresponding to MRP1 in membrane proteins prepared from both wild-type and P1150A mutant MRP1-transfected cells.
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ABCC1 p.Pro1150Ala 14722114:212:168
status: NEW214 In contrast, orthovanadate-induced trapping of 8-azido-[␣-32 P]ADP at 37 °C by the P1150A mutant was markedly reduced compared with wild-type MRP1 (Fig. 9B).
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ABCC1 p.Pro1150Ala 14722114:214:95
status: NEW226 ATP dependence of [3 H]E217betaG and [3 H]LTC4 transport by wild-type and P1150A mutant MRP1 proteins.
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ABCC1 p.Pro1150Ala 14722114:226:74
status: NEW227 Initial rates of [3 H]E217betaG uptake (A) and [3 H]LTC4 uptake (B) by wild-type MRP1 (f) and the P1150A mutant (Œ) were measured for 1 min in the presence of 10 different concentrations of ATP (2 M to 4 mM).
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ABCC1 p.Pro1150Ala 14722114:227:98
status: NEW231 8-Azido-[␣-32 P]ATP binding and orthovanadate-induced trapping of 8-azido-[␣-32 P]ADP by wild-type and P1150A mutant MRP1 proteins.
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ABCC1 p.Pro1150Ala 14722114:231:117
status: NEW280 The P1150A mutant showed a moderate but significant decrease in LTC4, GSH, and E13SO4 transport but, remarkably, also exhibited a substantial increase in E217betaG and MTX transport.
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ABCC1 p.Pro1150Ala 14722114:280:4
status: NEW281 The decrease in LTC4 transport caused by the P1150A mutation was associated with a 2.5-fold decrease in Vmax, whereas the increase in E217betaG transport was associated with a 4-fold increase in uptake affinity (decreased Km).
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ABCC1 p.Pro1150Ala 14722114:281:45
status: NEW282 These observations indicate that the structural change introduced by Ala substitution of Pro1150 in some way selectively enhances the apparent uptake affinity of MRP1 for the glucuronidated estrogen while at the same time diminishes the efficiency of LTC4 transport.
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ABCC1 p.Pro1150Ala 14722114:282:69
status: NEW283 Intriguingly, the P1150A mutation also affected the apparent ATP dependence of organic anion transport in a substrate-selective way.
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ABCC1 p.Pro1150Ala 14722114:283:18
status: NEW284 Thus, the P1150A mutation caused a 5-fold increase in apparent affinity (decreased Km) for ATP and 2-fold increase in Vmax when supporting E217betaG transport.
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ABCC1 p.Pro1150Ala 14722114:284:10
status: NEW285 However, for LTC4 transport, the Km(ATP) of P1150A was unchanged, and a 2-fold decrease in Vmax was observed.
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ABCC1 p.Pro1150Ala 14722114:285:44
status: NEW286 Furthermore, whereas the P1150A mutant appeared to bind azido-ATP as well as wild-type MRP1, it showed substantially reduced vanadate- and BeF-induced trapping of azido-ADP.
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ABCC1 p.Pro1150Ala 14722114:286:25
status: NEW289 This could contribute, at least in part, to the moderately reduced ability of the P1150A mutant to transport LTC4, because previous studies have indicated that ATP binding/ hydrolysis at NBD2 plays a dominant role in the transport of this substrate (46, 56).
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ABCC1 p.Pro1150Ala 14722114:289:82
status: NEW290 However, it is not immediately obvious how reduced ATP binding/hydrolysis activity at NBD2 can be reconciled with the enhanced ability of the P1150A mutant to transport E217betaG and MTX.
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ABCC1 p.Pro1150Ala 14722114:290:142
status: NEW291 One possibility is that ADP release from NBD2 of the P1150A mutant after hydrolysis is so rapid that the efficiency of vanadate-induced trapping of ADP is reduced, but this cannot explain the substrate-specific changes observed.
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ABCC1 p.Pro1150Ala 14722114:291:53
status: NEW294 MTX transport by the P1150A mutant was increased to an even greater extent than E217betaG transport.
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ABCC1 p.Pro1150Ala 14722114:294:21
status: NEW297 These structural differences are certain to affect the H-bonding capabilities of the two drugs, but whether or not they are related to the differing ability of the P1150A mutant to transport these two substrates awaits the availability of a more refined structure of MRP1.
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ABCC1 p.Pro1150Ala 14722114:297:164
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
1 We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C4 (LTC4) transport but increased 17beta-estradiol 17beta-D-glucuronide (E217betaG) and methotrexate (MTX) transport.
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ABCC1 p.Pro1150Ala 17494643:1:72
status: NEW2 Vanadate-induced trapping of [␣-32 P]8N3ADP 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.
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ABCC1 p.Pro1150Ala 17494643:2:58
status: NEW5 As observed for MRP1-Pro1150Ala, LTC4 transport by MRP2-Pro1158Ala was decreased.
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ABCC1 p.Pro1150Ala 17494643:5:21
status: NEW6 However, E217betaG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala.
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ABCC1 p.Pro1150Ala 17494643:6:127
status: NEW8 MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala.
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ABCC1 p.Pro1150Ala 17494643:8:89
status: NEW39 Compared with wild-type MRP1, MRP1-Pro1150Ala displayed decreased levels of LTC4, estrone sulfate, and GSH transport but substantially increased levels of E217betaG and methotrexate (MTX) transport.
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ABCC1 p.Pro1150Ala 17494643:39:35
status: NEW41 Although no difference in ATP binding was detected, the ability of MRP1-Pro1150Ala to trap 8N3ADP under hydrolytic conditions in the presence of sodium orthovanadate (but in the absence of substrate) was severely diminished.
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ABCC1 p.Pro1150Ala 17494643:41:72
status: NEW42 Because the ability to detect vanadate-inducible trapped [␣-32 P]8N3ADP complexes is often considered an indicator of the ATPase activity of a protein (Urbatsch et al., 1995), our observations suggested that either ATP hydrolysis by the MRP1-Pro1150Ala mutant was reduced or that the release of ADP may be enhanced (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:42:249
status: NEW43 It was also observed that although the apparent Km(ATP) for both wild-type MRP1 and MRP1-Pro1150Ala was the same during LTC4 transport, the Km(ATP) for the mutant transporter was reduced more than 4-fold during E217betaG transport.
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ABCC1 p.Pro1150Ala 17494643:43:89
status: NEW44 This change in ATP dependence suggests that E217betaG, but not LTC4, interacts with the MRP1-Pro1150Ala mutant in a way that increases its apparent affinity for ATP.
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ABCC1 p.Pro1150Ala 17494643:44:93
status: NEW45 The complexity of the MRP1-Pro1150Ala mutant phenotype, together with the high conservation of this Pro residue among ABCC family members (Fig. 1), has prompted us to investigate whether the corresponding residues in the MRP1 homologs, MRP2 and MRP3, are also functionally important.
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ABCC1 p.Pro1150Ala 17494643:45:27
status: NEW57 Generation of the MRP1-Pro1150Ala mutant has been described previously (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:57:23
status: NEW119 We showed previously that the MRP1-Pro1150Ala mutant was expressed in transfected HEK293T cells at levels comparable to those for wild-type MRP1 (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:119:35
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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ABCC1 p.Pro1150Ala 17494643:122:55
status: NEW129 As we reported previously, LTC4 transport by MRP1-Pro1150Ala was decreased by approximately 50% (Fig. 3A) (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:129:50
status: NEW138 On the other hand, E217betaG transport by MRP1-Pro1150Ala was significantly increased (2-fold) relative to wild-type MRP1 as expected (Fig. 3B) (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:138:47
status: NEW139 Also as expected for MRP1-Pro1150Ala, MTX transport was significantly increased by approximately ϳ3-fold (Fig. 3C) (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:139:26
status: NEW141 Protein expression levels of mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala and their corresponding wild-type proteins.
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ABCC1 p.Pro1150Ala 17494643:141:42
status: NEW148 Vesicular transport of 3 H-labeled organic anions by mutants MRP1-Pro1150Ala, MRP2-Pro1158Ala, and MRP3-Pro1147Ala.
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ABCC1 p.Pro1150Ala 17494643:148:66
status: NEW158 MTX transport by the MRP3-Pro1147Ala mutant was increased relative to that of wild-type MRP3 but to a lesser extent than was observed for MRP1-Pro1150Ala (1.5-fold versus 3-fold) (Fig. 3C).
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ABCC1 p.Pro1150Ala 17494643:158:143
status: NEW162 As shown previously, [3 H]LTC4 photolabeling of MRP1 was not affected by the Pro1150Ala mutation (Fig. 4A) (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:162:77
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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ABCC1 p.Pro1150Ala 17494643:165:15
status: NEW166 We previously showed that photolabeling of MRP1-Pro1150Ala by [␣-32 P]8N3ATP was comparable to that of wild-type MRP1 (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:166:48
status: NEW169 As reported previously, when MRP1-Pro1150Ala was exposed to vanadate (1 mM) and [␣-32 P]8N3ATP under hydrolytic conditions (37°C), the trapping of [␣-32 P]8N3ADP was very low compared with that of wild-type MRP1 (Fig. 6A) (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:169:34
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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ABCC1 p.Pro1150Ala 17494643:175:81
status: NEW186 The complex phenotype of the MRP1-Pro1150Ala mutant and the fact that Pro1150 is highly conserved in the ABCC subfamily suggested to us that this residue might serve an important functional role in other members of this class of ABC transporters (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:186:34
status: NEW188 For this reason, the consequences of replacing the Pro residues in MRP2 and MRP3 corresponding to MRP1-Pro1150 with Ala on the transport and nucleotide binding properties of the mutant proteins were examined.
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ABCC1 p.Pro1150Ala 17494643:188:103
status: NEW189 As described earlier, the MRP1-Pro1150Ala mutant showed substrate-selective changes in its transport activity (Koike et al., 2004).
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ABCC1 p.Pro1150Ala 17494643:189:31
status: NEW192 In contrast, the reduced LTC4 transport by MRP1-Pro1150Ala was associated with a decreased Vmax for LTC4 transport, whereas no change in Km(ATP) was observed (Koike et al., 2004).
X
ABCC1 p.Pro1150Ala 17494643:192:48
status: NEW197 As for MRP1-Pro1150Ala, the transport activities of the MRP2 and MRP3 Pro mutants were altered in a substrate-selective manner, demonstrating the conserved functional importance of a Pro residue at the NH2-proximal end of CL7.
X
ABCC1 p.Pro1150Ala 17494643:197:12
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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ABCC1 p.Pro1150Ala 17494643:208:38
status: NEW[hide] Role of proline 1150 in functional interactions be... Biochem Pharmacol. 2008 Apr 15;75(8):1659-69. Epub 2008 Feb 5. Letourneau IJ, Nakajima A, Deeley RG, Cole SP
Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter.
Biochem Pharmacol. 2008 Apr 15;75(8):1659-69. Epub 2008 Feb 5., 2008-04-15 [PMID:18336795]
Abstract [show]
The ATP-binding cassette multidrug resistance protein 1 (MRP1) mediates ATP-dependent cellular efflux of drugs and organic anions. We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E217betaG) and methotrexate transport as well as altered interactions with ATP. We have now further explored the functional importance of MRP1-Pro1150 at the interface of transmembrane helix 15 and cytoplasmic loop 7 (CL7) by replacing it with Gly, Ile, Leu and Val. All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [gamma32P]8N3ATP photolabeling. They also displayed very low levels of substrate-independent vanadate-induced trapping of [alpha32P]8N3ADP. To better understand the relationship between the altered nucleotide interactions and transport activity of these mutants, [alpha32P]8N3ADP trapping experiments were performed under different conditions. Unlike leukotriene C4, E217betaG decreased [alpha32P]8N3ADP trapping by both wild-type and mutant MRP1. [alpha32P]8N3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni2+ instead of Mg2+, and by decreasing temperature; however, the transport properties of the mutant remained unchanged. We conclude that the reduced [alpha32P]8N3ADP trapping associated with loss of Pro1150, or the presence of E217betaG, is due to enhanced ADP release following ATP hydrolysis rather than a reduction in ATP hydrolysis itself. We hypothesize that loss of Pro1150 alters the role of CL7 as a coupling helix that mediates signaling between the nucleotide binding domains and some substrate binding sites in the membrane spanning domains of MRP1.
Comments [show]
None has been submitted yet.
No. Sentence Comment
4 We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E217bG) and methotrexate transport as well as altered interactions with ATP.
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ABCC1 p.Pro1150Ala 18336795:4:39
status: NEW6 All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [g32 P]8N3ATP photolabeling.
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ABCC1 p.Pro1150Ala 18336795:6:55
status: NEW10 [a32 P]8N3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni2+ instead of Mg2+ , and by decreasing temperature; however, the transport properties of the mutant remained unchanged.
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ABCC1 p.Pro1150Ala 18336795:10:31
status: NEW27 Compared to wild-type MRP1, the Pro1150Ala mutant displayed decreased levels of LTC4, estrone sulfate, and GSH transport but substantially increased levels of E217bG and methotrexate (MTX) transport.
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ABCC1 p.Pro1150Ala 18336795:27:32
status: NEW28 The increased E217bG transport by MRP1-Pro1150Ala was associated with a 5-fold decrease in apparent Km(E217bG) and 4-fold decrease in Km(ATP) during E217bG transport.
X
ABCC1 p.Pro1150Ala 18336795:28:39
status: NEW29 However, the apparent Km(ATP) values for wild-type MRP1 and MRP1-Pro1150Ala were the same during LTC4 transport.
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ABCC1 p.Pro1150Ala 18336795:29:65
status: NEW30 The interaction of the MRP1-Pro1150Ala mutant with nucleotide was further investigated using the 32 P-labeled photoaffinity ligand 8N3ATP.
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ABCC1 p.Pro1150Ala 18336795:30:28
status: NEW32 In the case of MRP1-Pro1150Ala, vanadate-induced trapping of [a32 P]8N3ADP was greatly diminished relative to wild-type MRP1, while photolabeling of this mutant with [g32 P]8N3ATP under nonhydrolytic conditions was unchanged [11].
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ABCC1 p.Pro1150Ala 18336795:32:20
status: NEW49 Site-directed mutagenesis The generation of the MRP1-Pro1150Ala mutant expression construct has been described previously [11].
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ABCC1 p.Pro1150Ala 18336795:49:53
status: NEW76 Statistical comparisons of the IC50 values for wild-type MRP1 and MRP1-Pro1150Ala were carried out using a paired Student t-test and considered significant when p < 0.05.
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ABCC1 p.Pro1150Ala 18336795:76:71
status: NEW95 Mutation of Pro1150 affects the trypsin sensitivity of MRP1 To determine whether the functional impact of the MRP1-Pro1150 mutation was associated with changes in MRP1 structure, limited trypsin digests of wild-type MRP1 and the MRP1-Pro1150Ala mutant were performed side-by-side and the fragments probed by immunoblotting with MAbs against the NH2-proximal (N1, N3) (MAb MRPr1) and COOH-proximal (C1, C2) (MAb MRPm6) halves of MRP1 (Fig. 1A).
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ABCC1 p.Pro1150Ala 18336795:95:234
status: NEW96 As shown in Fig. 1B and C, the Pro1150Ala mutant was more resistant to trypsin digestion than wild-type MRP1 as evident from the persistence of the full-length protein at high trypsin:protein ratios.
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ABCC1 p.Pro1150Ala 18336795:96:31
status: NEW102 Immunoblots of the membrane vesicles prepared after transient expression in HEK293T cells revealed that none of the four mutations affected levels of MRP1 expression relative to wild-type MRP1 b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 5 9 - 1 6 6 9 1661 as shown previously for the Pro1150Ala mutant (Fig. 2A) [11,13].
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ABCC1 p.Pro1150Ala 18336795:102:307
status: NEW105 As shown in Fig. 2B-D, the transport properties of all four mutants were similar to that described previously for MRP1-Pro1150Ala [11,13], viz., the mutants exhibited an approximately 50% decrease in LTC4 transport (Fig. 2B), a 2-fold increase in E217bG transport (Fig. 2C), and a 3-fold increase in MTX transport (Fig. 2D).
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ABCC1 p.Pro1150Ala 18336795:105:119
status: NEW108 As observed previously, [3 H]LTC4 photolabeling of the Pro1150Ala mutant was similar to that of wild-type MRP1 (Fig. 3A) [11].
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ABCC1 p.Pro1150Ala 18336795:108:55
status: NEW110 We next examined whether the binding of other MRP1 substrates (e.g. E217bG and MTX) was affected by Ala substitution of Pro1150 .
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ABCC1 p.Pro1150Ala 18336795:110:100
status: NEW112 As shown in Fig. 3B, E217bG inhibited [3 H]LTC4 photolabeling of wild-type MRP1 and the MRP1-Pro1150Ala mutant in a comparable concentration dependent fashion.
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ABCC1 p.Pro1150Ala 18336795:112:93
status: NEW113 [3 H]LTC4 photolabeling of wild-type and Pro1150Ala mutant MRP1 was also similarly inhibited by MTX (Fig. 3C).
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ABCC1 p.Pro1150Ala 18336795:113:41
status: NEW114 These results indicate that binding of LTC4, E217bG and MTX to wild-type MRP1 and the MRP1-Pro1150Ala mutant is similar, and thus suggest that the changes observed in the transport of these substrates are not due to changes in their initial binding to MRP1.
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ABCC1 p.Pro1150Ala 18336795:114:91
status: NEW115 Comparable experiments were not carried out for the other Pro1150 mutants because their transport activities were so similar to those of Pro1150Ala and accordingly, would be expected to exhibit a similar pattern of substrate competition.
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ABCC1 p.Pro1150Ala 18336795:115:137
status: NEW118 To test this, E217bG transport by the MRP1-Pro1150Ala mutant was examined in the presence of MK571, S-decyl-GSH, BAY u9773 and LY465803.
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ABCC1 p.Pro1150Ala 18336795:118:43
status: NEW121 As shown in Fig. 4, MK571, S-decyl-GSH, BAY u9773 and LY465803 inhibited MRP1-mediated E217bG transport with IC50 values Fig. 1 - Limited trypsin digestion of wild-type MRP1 and Pro1150Ala mutant MRP1.
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ABCC1 p.Pro1150Ala 18336795:121:178
status: NEW127 b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 5 9 - 1 6 6 91662 which were similar for both wild-type MRP1 and the MRP1-Pro1150Ala mutant ( p > 0.05).
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ABCC1 p.Pro1150Ala 18336795:127:141
status: NEW128 The effect of the four modulators on E217bG transport of the other Pro1150 mutants was not tested because their transport activities were so similar to those of Pro1150Ala that it would be reasonable to expect them to exhibit similar patterns of inhibitor sensitivity.
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ABCC1 p.Pro1150Ala 18336795:128:161
status: NEW130 Pro1150 mutants interact similarly with 32 P-labeled nucleotide We have reported previously that MRP1-Pro1150Ala and wild-type MRP1 bind similar levels of 8N3ATP but vanadate-induced trapping of 8N3ADP by the mutant is greatly reduced [11].
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ABCC1 p.Pro1150Ala 18336795:130:102
status: NEW145 Even when photolabeling was carried out with a broader range of [g32 P]8N3ATP concentrations, no differences in photolabeling of the Pro1150Ala mutant and wild-type MRP1 were observed (results not shown).
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ABCC1 p.Pro1150Ala 18336795:145:133
status: NEW146 When nucleotide binding was determined under hydrolysis conditions (37 8C) and in the presence of vanadate, the Pro1150Ala mutant showed a substantial reduction (approximately 70%) in levels of trapped [a32 P]8N3ADP as expected [11].
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ABCC1 p.Pro1150Ala 18336795:146:112
status: NEW149 However, as observed when ATP was used, E217bG uptake by MRP1-Pro1150Ala in the presence of 8N3ATP was still 2-fold higher than uptake by wild-type MRP1 (results not shown).
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ABCC1 p.Pro1150Ala 18336795:149:62
status: NEW150 To exclude the possibility that diminished interaction between the mutant transporter and vanadate was responsible for the decreased [a32 P]8N3ADP trapping observed, the effect of different concentrations of vanadate on E217bG transport by the Pro1150Ala mutant was compared with wild-type MRP1.
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ABCC1 p.Pro1150Ala 18336795:150:244
status: NEW151 The IC50 for vanadate-mediated inhibition of E217bG transport was $0.5 mM for wild-type MRP1 and $1 mM for the Pro1150Ala mutant (results not shown).
X
ABCC1 p.Pro1150Ala 18336795:151:111
status: NEW154 E217bG decreases vanadate-induced [a32 P]8N3ADP trapping by wild-type and Pro1150Ala mutant MRP1 It is generally believed that the presence of substrates can stimulate the ATPase activity of ABC transporters and can thereby enhance levels of vanadate-induced trapping of ADP.
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ABCC1 p.Pro1150Ala 18336795:154:74
status: NEW156 Because E217bG transport by the MRP1-Pro1150Ala mutant was substantially increased, it was of interest to determine if ADP trapping by the wild-type and mutant proteins might be differentially influenced by this substrate.
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ABCC1 p.Pro1150Ala 18336795:156:37
status: NEW157 Consequently, [a32 P]8N3ADP trapping experiments with the Pro1150Ala mutant were repeated in the presence of Fig. 4 - Effect of MRP1 modulators on E217bG transport by wild-type MRP1 and MRP1-Pro1150Ala.
X
ABCC1 p.Pro1150Ala 18336795:157:58
status: NEWX
ABCC1 p.Pro1150Ala 18336795:157:191
status: NEW160 Open symbols represent data obtained with vesicles prepared from cells expressing wild-type MRP1 and filled symbols represent data obtained with vesicles prepared from cells expressing the MRP1-Pro1150Ala mutant.
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ABCC1 p.Pro1150Ala 18336795:160:194
status: NEW172 Trapping of [a32 P]8N3ADP by the MRP1-Pro1150Ala mutant was also decreased in the presence of E217bG.
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ABCC1 p.Pro1150Ala 18336795:172:38
status: NEW175 Release of 8N3ADP from MRP1-Pro1150Ala is enhanced relative to wild-type MRP1 While the decreased trapping of 8N3ADP by MRP1-Pro1150Ala could be due to reduced ATP hydrolysis, it might also be due to enhanced release of the dinucleotide.
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ABCC1 p.Pro1150Ala 18336795:175:28
status: NEWX
ABCC1 p.Pro1150Ala 18336795:175:125
status: NEW178 At 23 8C, lower levels of vanadate-trapped [a32 P]8N3ADP by wild-type MRP1 were observed; however, at 37 8C, the amount of [a32 P]8N3ADP trapped by the Pro1150Ala mutant was 40% that of wild-type MRP1 while at 23 8C, the amount trapped increased to 90% of wild-type MRP1.
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ABCC1 p.Pro1150Ala 18336795:178:152
status: NEW179 To determine if the increased trapping of [a32 P]8N3ADP at 23 8C by MRP1-Pro1150Ala was associated with any changes in its transport activity relative to wild-type MRP1, vesicular transport assays were performed at both 23 and 37 8C.
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ABCC1 p.Pro1150Ala 18336795:179:73
status: NEW180 As shown in Fig. 7B, E217bG transport by the Pro1150Ala mutant was 2-to 3-fold higher than wild-type MRP1 at both temperatures.
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ABCC1 p.Pro1150Ala 18336795:180:45
status: NEW181 Thus, no differences in relative transport activity were observed at 23 8C despite comparable levels of [a32 P]8N3ADP trapping (and implied ATP hydrolysis) by wild-type and Pro1150Ala mutant MRP1 at this lower temperature.
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ABCC1 p.Pro1150Ala 18336795:181:173
status: NEW182 The possibility that reduced vanadate-induced trapping by MRP1-Pro1150Ala at 37 8C was due to differences in the ability of wild-type MRP1 and MRP1-Pro1150Ala mutant to interact with divalent metal cations was also explored.
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ABCC1 p.Pro1150Ala 18336795:182:63
status: NEWX
ABCC1 p.Pro1150Ala 18336795:182:148
status: NEW183 Thus, Mn2+ , Co2+ , Ni2+ , Zn2+ and Cd2+ were tested for their ability to support vanadate-induced trapping of [a32 P]8N3ADP by the wild-type and MRP1-Pro1150Ala mutant proteins, and compared to the physiological cation Mg2+ .
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ABCC1 p.Pro1150Ala 18336795:183:151
status: NEW187 With most divalent cations that supported [a32 P]8N3ADP trapping, the MRP1-Pro1150Ala mutant still trapped significantly less dinucleotide than wild-type MRP1 (!65% decrease).
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ABCC1 p.Pro1150Ala 18336795:187:75
status: NEW188 Interestingly, when Ni2+ was used, the difference in trapping levels between the mutant and wild-type MRP1 proteins was reduced, with the trapping signal for MRP1-Pro1150Ala increasing from approximately 40% of wild-type MRP1 in the presence of Mg2+ to approximately 70% in the presence of Ni2+ (Fig. 7A, bottom panel).
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ABCC1 p.Pro1150Ala 18336795:188:163
status: NEW189 E217bG vesicular uptake measured under these conditions was approximately 20-fold lower in the presence of Ni2+ compared to Mg2+ , but the >2-fold difference in E217bG transport activity between wild-type MRP1 and the MRP1-Pro1150Ala mutant was still observed (Fig. 7B).
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ABCC1 p.Pro1150Ala 18336795:189:223
status: NEW197 Thus, replacement of Pro1150 with alanine is a non-conservative substitution since the small hydrophobic side chain of alanine can support the formation of a-helices although it does not necessarily result in the straightening of the a-helix [26,28,29].
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ABCC1 p.Pro1150Ala 18336795:197:21
status: NEW199 In contrast, substitution of Pro1150 with glycine may be considered a conservative substitution because glycine, like Fig. 6 - Effect of E217bG on vanadate-induced trapping of [a32 P]8N3ADP by wild-type and Pro1150Ala mutant MRP1.
X
ABCC1 p.Pro1150Ala 18336795:199:207
status: NEW201 Relative levels of vanadate-induced [a32 P]8N3ADP trapping are indicated in italics and have been corrected where necessary to take into account differences in expression of the Pro1150Ala mutant relative to wild-type MRP1.
X
ABCC1 p.Pro1150Ala 18336795:201:178
status: NEW204 Although the tryptic digestion pattern of the Pro1150Ala mutant differed from that of the wild-type protein, indicating that loss of Pro1150 does introduce some change in the structure of MRP1, we observed no differences in expression levels of the various MRP1-Pro1150 mutants.
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ABCC1 p.Pro1150Ala 18336795:204:46
status: NEW210 Similarly, inhibition of [3 H]LTC4 photolabeling by E217bG and MTX was comparable for the wild-type and MRP1-Pro1150Ala proteins.
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ABCC1 p.Pro1150Ala 18336795:210:109
status: NEW211 Therefore, it may be concluded that the substrate selective increases in E217bG and MTX transport activities are not due to substantial changes in initial Fig. 7 - Effects of temperature and divalent cations on vanadate-induced [a32 P]8N3ADP trapping and E217bG transport by wild-type and Pro1150Ala mutant MRP1.
X
ABCC1 p.Pro1150Ala 18336795:211:289
status: NEW216 HEK (black bars), wild-type-MRP1 (open bars) and MRP1-Pro1150Ala (grey bars).
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ABCC1 p.Pro1150Ala 18336795:216:54
status: NEW220 Furthermore, despite the marked differences in their chemical structures, specificity and mode of inhibitory action, the unchanged IC50 values for the MRP1 modulators MK571, BAY u9773, S-decylGSH and LY465803 also suggest that recognition of these compounds is unaffected by the MRP1-Pro1150Ala mutation.
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ABCC1 p.Pro1150Ala 18336795:220:284
status: NEW229 In contrast to LTC4, however, we now find that E217bG decreases [a32 P]8N3ADP trapping by both wild-type MRP1 and the Pro1150Ala mutant.
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ABCC1 p.Pro1150Ala 18336795:229:118
status: NEW231 Trapping of [a32 P]8N3ADP by the MRP1-Pro1150Ala mutant was also decreased in the presence of E217bG.
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ABCC1 p.Pro1150Ala 18336795:231:38
status: NEW235 To explain the decreased vanadate-induced [a32 P]8N3ADP trapping by the MRP1-Pro1150Ala mutant, we hypothesized that rather than decreasing ATP hydrolysis, the mutation was enhancing the release of ADP following ATP hydrolysis, most of which is known to occur at NBD2 [9,33].
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ABCC1 p.Pro1150Ala 18336795:235:77
status: NEW238 We found that by carrying out the reactions at 23 8C instead of 37 8C (which would presumably increase the occupancy time of 8N3ADP in NBD2), [a32 P]8N3ADP trapping by MRP1-Pro1150Ala could be increased to levels comparable to that of wild-type MRP1 (Fig. 7).
X
ABCC1 p.Pro1150Ala 18336795:238:173
status: NEW244 Although Co2+ and Mn2+ supported vanadate-induced [a32 P]8N3ADP trapping by wild-type MRP1 to a similar or higher level than Ni2+ , they were unable to increase [a32 P]8N3ADP trapping by the MRP1-Pro1150Ala mutant to the same extent as Ni2+ (result not shown).
X
ABCC1 p.Pro1150Ala 18336795:244:196
status: NEW246 Overall, our data clearly indicate that the changes in the transport activities of MRP1-Pro1150Ala and other Pro1150 mutants are not tightly linked to the reduced vanadate-induced 8N3ADP trapping observed.
X
ABCC1 p.Pro1150Ala 18336795:246:88
status: NEW[hide] Structural and functional properties of human mult... Curr Med Chem. 2011;18(3):439-81. He SM, Li R, Kanwar JR, Zhou SF
Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1).
Curr Med Chem. 2011;18(3):439-81., [PMID:21143116]
Abstract [show]
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.
Comments [show]
None has been submitted yet.
No. Sentence Comment
755 Compared with wild-type MRP1/ABCC1, the Pro1150Ala mutant showed decreased LTC4, estrone sulfate, and GSH transport but substantially increased E217 G and MTX transport [345].
X
ABCC1 p.Pro1150Ala 21143116:755:40
status: NEW757 In the case of Pro1150Ala mutant, vanadate-induced trapping of [ 32 P]8N3ADP was greatly diminished relative to wild-type MRP1, while photolabeling of this mutant with [ 32 P]8N3ATP under nonhydrolytic conditions remained unchanged [345].
X
ABCC1 p.Pro1150Ala 21143116:757:15
status: NEW758 The Pro1150Gly, Pro1150Ile, Pro1150Leu and Pro1150Val mutants exhibited a phenotype similar to the Pro1150Ala mutant with respect to organic anion transport and [ 32 P]8N3ATP photolabeling [347].
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ABCC1 p.Pro1150Ala 21143116:758:99
status: NEW[hide] Multidrug resistance protein 1 (MRP1, ABCC1), a "m... J Biol Chem. 2014 Nov 7;289(45):30880-8. doi: 10.1074/jbc.R114.609248. Epub 2014 Oct 3. Cole SP
Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATP-binding cassette (ABC) transporter.
J Biol Chem. 2014 Nov 7;289(45):30880-8. doi: 10.1074/jbc.R114.609248. Epub 2014 Oct 3., [PMID:25281745]
Abstract [show]
The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases.
Comments [show]
None has been submitted yet.
No. Sentence Comment
69 When Pro1150 is replaced by Ala (or Leu or Val), the mutant protein exhibits substantially increased levels of methotrexate and E217betaG transport, due to a change in Kmapp (39).
X
ABCC1 p.Pro1150Ala 25281745:69:5
status: NEW70 This increase is substrate-selective, however, because the transport of other OAs (e.g. LTC4) by the P1150A mutant is unchanged.
X
ABCC1 p.Pro1150Ala 25281745:70:101
status: NEW