ABCC3 p.Thr1237Ala
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (85%), E: D (85%), F: D (80%), G: D (75%), H: D (80%), I: D (75%), K: D (91%), L: D (80%), M: D (66%), N: D (63%), P: D (91%), Q: D (80%), R: D (91%), S: N (61%), V: D (75%), W: D (85%), Y: D (80%), |
| 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, R: D, S: D, V: D, W: D, Y: D, |
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[hide] Characterization of the role of polar amino acid r... Biochemistry. 2003 Aug 26;42(33):9989-10000. Zhang DW, Gu HM, Vasa M, Muredda M, Cole SP, Deeley RG
Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3.
Biochemistry. 2003 Aug 26;42(33):9989-10000., [PMID:12924948]
Abstract [show]
Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.
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No. Sentence Comment
9 In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates.
X
ABCC3 p.Thr1237Ala 12924948:9:22
status: NEW75 They are as follows: S1229A (5'-GGG CTG GTG GGG CTA GCT GTG TCC TAC TCC-3'), S1231A (5'-GC CTT TCT GTG GCC TAC TCC CTG CAG GTG ACA-3'), Y1232F (5'-T TCT GTG TCC TTC TCC TTA CAG GTG ACA TTT G-3'), S1233A (5'-CT GTG TCC TAC GCC CTG CAG GTG ACA TTT G-3'), Q1235A (5'-G TCC TAC TCC TTG GCG GTG ACA TTT GCT C-3'), T1237A (5'-CC TTG CAG GTG GCA TTC GCT CTG AAC TGG-3'), T1237S (5'-CC TTG CAG GTG TCC TTC GCT CTG AAC TGG-3'), T1237G (5'-CC TTG CAG GTG GGA TTC GCT CTG AAC TGG-3'), T1237L (5'-CC TTG CAG GTG CTA TTC GCT CTG AAC TGG-3'), and N1241A (5'-GTG ACA TTT GCG CTA GCC TGG ATG ATA C-3').
X
ABCC3 p.Thr1237Ala 12924948:75:309
status: NEW139 In contrast, conversion of Thr1237 to Ala resulted in a significant (~2.0-fold) increase in transport activity.
X
ABCC3 p.Thr1237Ala 12924948:139:27
status: NEW147 Similar to the results obtained with E217βG as a substrate, substitution of Thr1237 with Ala resulted in a mutant protein that displayed an enhanced transport activity of the drug.
X
ABCC3 p.Thr1237Ala 12924948:147:82
status: NEW151 In contrast, mutations Q1235A and T1237A increased the ability of MRP3 to transport the bile salt approximately 1.5-and 3-fold, respectively.
X
ABCC3 p.Thr1237Ala 12924948:151:34
status: NEW154 We have shown that mutation T1237A increased the ability of MRP3 to transport both E217βG and taurocholate.
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ABCC3 p.Thr1237Ala 12924948:154:28
status: NEW155 Like mutation T1237A, replacement of Gln1235 with Ala also increased taurocholate uptake.
X
ABCC3 p.Thr1237Ala 12924948:155:14
status: NEW160 The results suggest that mutations Q1235A and T1237A affect a step in the transport process after initial binding of this substrate.
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ABCC3 p.Thr1237Ala 12924948:160:46
status: NEW161 The effects of mutations Q1235A and T1237A on kinetic parameters of E217βG transport were also examined (Figure 7B, Table 1).
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ABCC3 p.Thr1237Ala 12924948:161:36
status: NEW180 Interestingly, converting Thr1237 to Ala decreased Km values for E217βG transport by approximately 5-fold (Km ) 6 µM for MRP1T1237A ).
X
ABCC3 p.Thr1237Ala 12924948:180:26
status: NEW182 Thus, in contrast to the results obtained with taurocholate, these findings demonstrated that the increase in E217βG transport observed with the T1237A mutation at subsaturating concentrations was attributable to increased affinity of the protein for this substrate, which is associated with a decrease in Vmax.
X
ABCC3 p.Thr1237Ala 12924948:182:151
status: NEW187 Interestingly, conversion of Thr1237 to Ala resulted in a mutant protein with enhanced resistance to VP-16 (3-fold).
X
ABCC3 p.Thr1237Ala 12924948:187:29
status: NEW188 Thus, on the basis of the substrates tested in this study, mutations S1229A, S1231A, Q1235A, and N1241A affected substrate specificity of MRP3, whereas mutations Y1232F, S1233A, and T1237A influenced the overall activity of the protein.
X
ABCC3 p.Thr1237Ala 12924948:188:182
status: NEW190 Since replacement of Thr1237 with Ala resulted in a mutant protein with an enhanced capacity to confer VP-16 resistance, we also mutated this residue to Gly, Ser, and Leu.
X
ABCC3 p.Thr1237Ala 12924948:190:21
status: NEW193 As shown in Table 2, mutation T1237G, like T1237A, significantly FIGURE 4: ATP-dependent [3H]E217βG uptake by membrane vesicles prepared from HEK293 cells stably transfected with wild-type or mutant MRP3. Panel A: Relative protein expression levels of wild-type and mutant MRP3 proteins in stably transfected HEK293 cells.
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ABCC3 p.Thr1237Ala 12924948:193:43
status: NEW211 In addition to the effect on VP-16 resistance, substitution of Thr1237 with Ala increased the capacity of MRP3 to transport methotrexate, E217βG, and taurocholate.
X
ABCC3 p.Thr1237Ala 12924948:211:63
status: NEW213 Replacement of Thr1237 with Gly, like mutation T1237A, dramatically increased the ability of MRP3 to transport methotrexate (Figure 8B), E217βG (Figure 8C), and taurocholate (Figure 8D).
X
ABCC3 p.Thr1237Ala 12924948:213:47
status: NEW248 In MRP3, elimination of the hydrogen-bonding capability of Tyr1232 and Ser1233 negatively affected all functions, while mutation of Thr1237 to Ala had a positive effect.
X
ABCC3 p.Thr1237Ala 12924948:248:132
status: NEW288 In addition, conversion of polar residue Thr1237 to either Ala or Gly markedly increased the ability to confer VP-16 resistance and to transport E217βG, taurocholate, and methotrexate, while mutation to a bulkier and more hydrophobic Leu residue resulted in only a moderate increase in transport of all three substrates.
X
ABCC3 p.Thr1237Ala 12924948:288:41
status: NEW292 Interestingly, although mutation T1237A increased the ability of MRP3 to transport both taurocholate and E217βG, the mutation resulted in a major increase in Vmax for taurocholate uptake, without significantly affecting Km values.
X
ABCC3 p.Thr1237Ala 12924948:292:33
status: NEW295 If so, the fact that the T1237A mutations increase the affinity of the protein for E217βG suggests that a single amino acid may contribute both to the high-affinity binding of one substrate and the low-affinity binding of another.
X
ABCC3 p.Thr1237Ala 12924948:295:25
status: NEW