ABCC1 p.Asp1183Glu
| Predicted by SNAP2: | A: D (63%), C: D (63%), E: D (63%), F: D (75%), G: D (71%), H: D (75%), I: D (75%), K: D (80%), L: D (80%), M: D (75%), N: D (53%), P: D (85%), Q: D (66%), R: D (75%), S: D (59%), T: D (66%), V: D (75%), W: D (80%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: 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, T: D, V: D, W: D, Y: D, |
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[hide] Multiple roles of charged amino acids in cytoplasm... Mol Pharmacol. 2009 Feb;75(2):397-406. Epub 2008 Nov 17. Conseil G, Rothnie AJ, Deeley RG, Cole SP
Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1).
Mol Pharmacol. 2009 Feb;75(2):397-406. Epub 2008 Nov 17., [PMID:19015228]
Abstract [show]
Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.
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No. Sentence Comment
7 Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1.
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ABCC1 p.Asp1183Glu 19015228:7:19
status: NEW59 Mutations were first generated in the pGEM-3Z-XmaI/MRP1 plasmid according to the manufacturer`s instructions with the following mutagenic primers (Integrated DNA Technologies, Inc., Coralville, IA), which also introduced an additional restriction site (substituted nucleotides are underlined): E1144A (5Ј-G AAG CGC CTC GCG TCG GTC AGC-3Ј); R1166A (5Ј C AGC GTC ATT GCA GCA TTC GAG GAG CAG-3Ј); R1166K (5Ј C AGC GTC ATT AAG GCC TTC GAG G-3Ј); 1169 EEQE-1169 AAQA (5Ј-C ATT CGA GCC TTC GCG GCA CAG GCA CGC TTC ATC C-3Ј); R1173A (5Ј-C GAG GAG CAG GAG GCA TTC ATC CAC CAG AG-3Ј); D1179A (5Ј-C CAC CAG AGT GCC CTT AAG GTG GAC G-3Ј), K1181A (5Ј-G AGT GAC CTG GCA GTC GAC GAG AAC C-3Ј); D1183A (5Ј-CTG AAG GTG GCC GAG AAC CAG-3Ј); D1183E (5Ј- CTG AAG GTG GAA GAG AAC CAG-3Ј); and E1184A (5Ј-GT GAC CTG AAG GTA GAC GCG AAC CAG AAG GCC-3Ј).
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ABCC1 p.Asp1183Glu 19015228:59:820
status: NEW128 To determine whether it was the charge or other property of the amino acid at positions 1166 and 1183 that was critical for efficient plasma membrane expression of MRP1, same-charge mutants of Arg1166 (R1166K) and Asp1183 (D1183E) were created and again expressed in HEK cells.
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ABCC1 p.Asp1183Glu 19015228:128:223
status: NEW129 At 37°C, the MRP1 expression levels in whole-cell lysates were increased to 50% of wild-type MRP1 levels for the D1183E mutant, and for the R1166K mutant, expression was comparable with that of wild-type MRP1 (Fig. 2B).
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ABCC1 p.Asp1183Glu 19015228:129:118
status: NEW130 Transport Activity of R1166K and D1183E Mutant MRP1 Proteins.
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ABCC1 p.Asp1183Glu 19015228:130:33
status: NEW131 To characterize the transport properties of the same-charge R1166K and D1183E mutants, membrane vesicles were prepared from transfected HEK cells.
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ABCC1 p.Asp1183Glu 19015228:131:71
status: NEW134 Transport activity of the D1183E mutant was not assessed because its very low expression levels relative to wild-type MRP1 made measurements unreliable (Fig. 3A).
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ABCC1 p.Asp1183Glu 19015228:134:26
status: NEW144 A, representative immunoblot of membrane vesicles (1 g of protein per lane) prepared from HEK 293T cells transfected with wild-type (WT), R1166K, and D1183E cDNA expression vectors. MRP1 proteins were detected with mAb QCRL-1 and membrane vesicles from untransfected cells were included as a negative control.
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ABCC1 p.Asp1183Glu 19015228:144:158
status: NEW201 Our observation that the same-charge D1183E mutant was as poorly expressed at the plasma membrane as the neutrally substituted D1183A is somewhat unexpected, because previously identified expression impaired mutants involving charged residues have typically not exhibited this property (Haimeur et al., 2004; Situ et al., 2004).
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ABCC1 p.Asp1183Glu 19015228:201:37
status: NEW208 In contrast to D1183E, the same charge mutant of Arg1166 , R1166K, was expressed in membrane vesicles at levels that approached 50% those of wild-type MRP1, and the relative levels of LTC4 and E217betaG transport activity of this mutant were comparable with those of wild-type MRP1 after normalization for differences in protein expression levels (Fig. 3).
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ABCC1 p.Asp1183Glu 19015228:208:15
status: NEW