ABCC1 p.Cys43Ala
| Predicted by SNAP2: | A: D (80%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (59%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: 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, T: D, V: D, W: D, Y: D, |
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[hide] Functional and structural consequences of cysteine... Biochemistry. 2003 May 13;42(18):5214-24. Leslie EM, Letourneau IJ, Deeley RG, Cole SP
Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1).
Biochemistry. 2003 May 13;42(18):5214-24., 2003-05-13 [PMID:12731862]
Abstract [show]
The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.
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No. Sentence Comment
54 Mutagenesis was performed according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined and introduced or lost restriction sites are in italics) as follows: Cys43Ala (5'-G TGG GTG CCT GCT TTT TAC CTC TGG GCC-3'), Cys43Ser (5'-G TGG GTG CCT TCT TTT TAC CTC-3'), Cys49Ala (5'-C CTC TGG GCC GCA TTC CCC TTC TAC-3') (BsmI), Cys49Ser (5'-C CTC TGG GCC TCT TTC CCC TTC-3'), Cys85Ala (5'-G TGG ATC GTC GCG TGG GCA GAC C-3') (BstUI), Cys85Ser (5'-G TGG ATC GTC AGC TGG GCA GAC C-3'), Cys148Ala (5'-GTA GCC CTA GTG GCT GCC CTA GCC-3') (BglI), Cys148Ser (5'-GTA GCC CTA GTG TCT GCC CTA GCC-3'), Cys190Ala (5'-C GTC TTG TCC GCA TTC TCA GAT CGC-3') (BsmI), Cys190Ser (5'-C GTC TTG TCC TCT TTC TCA GAT CG-3'), Cys208Ala (5'-C CCT AAT CCC GCG CCA GAG TCC AG-3') (BstUI), Cys208Ser (5'-C CCT AAT CCC AGC CCA GAG TCC-3'), Cys265Ala (5'-GTA AAG AAC TGG AAG AAG GAA GCC GCG AAG ACT AGG AAG CAG-3') (BpiI), and Cys265Ser (5'- GG AAG AAG GAA TCC GCC AAG ACT AG-3') (BsmI).
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ABCC1 p.Cys43Ala 12731862:54:223
status: NEW110 Those exhibiting minor disruption of plasma membrane trafficking included cell lines expressing four CysfAla mutants [Cys43Ala (Figure 3C), Cys85Ala (Figure 3G), Cys190Ala (Figure 3K), and Cys208Ala (Figure 3M)] and four CysfSer mutants [Cys49Ser (Figure 3F), Cys85Ser (Figure 3H), Cys190Ser (Figure 3L), and Cys208Ser (Figure 3N)].
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ABCC1 p.Cys43Ala 12731862:110:118
status: NEW111 Three of the mutant cell lines with impaired plasma membrane trafficking of MRP1 exhibited filament-like staining (Cys43Ala, Cys43Ser, and Cys49Ser) (Figure 3C,D,F) while the cell lines expressing Cys265Ala and Cys265Ser MRP1 showed a stippled-like staining pattern.
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ABCC1 p.Cys43Ala 12731862:111:115
status: NEW130 Table 1: Detection of Tryptic Fragments N1 and N2 of MRP1 in Membranes Prepared from HeLa Cells Stably Expressing CysfAla and CysfSer MRP1 Mutantsa trypsin:protein ratio (w:w)transfected HeLa cell line N1 detected N2 detected WT-MRP1 1:10 000 1:1000 C43A-MRP1 1:100 1:100 C43S-MRP1 1:10 000 1:1000 C49A-MRP1 1:250 1:100 C49S-MRP1 1:10 000 1:500 C85A-MRP1 1:10 000 1:1000 C85S-MRP1 1:1000 1:250 C148A-MRP1 1:250 1:250 C148S-MRP1 1:1000 1:500 C190A-MRP1 1:1000 1:1000 C190S-MRP1 1:1000 1:250 C208A-MRP1 1:10 000 1:250 C208S-MRP1 1:10 000 1:500 C265A-MRP1 1:250 1:10 C265S-MRP1 1:1000 1:250 a The data shown represent a summary of the limited trypsin digests shown in Figures 4 and 5.
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ABCC1 p.Cys43Ala 12731862:130:250
status: NEW134 Trypsinolysis of the four remaining Ala-substituted Cys mutants (Cys148Ala, Cys49Ala, Cys43Ala, and Cys265Ala) showed that they were also quite resistant to cleavage by this enzyme.
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ABCC1 p.Cys43Ala 12731862:134:86
status: NEW135 For example, the N1 and N2 fragments of Cys148Ala-MRP1 did not appear until a trypsin:protein ratio of 1:250 while the N1 and N2 fragments of Cys49Ala-MRP1 appeared at ratios of 1:250 and 1:100, respectively. The Cys43Ala-MRP1 mutant was highly resistant to trypsinolysis with the appearance of the N1 band not occurring until the trypsin:protein ratio was 1:100, the same ratio at which the N2 band appeared.
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ABCC1 p.Cys43Ala 12731862:135:213
status: NEW136 The Cys265Ala MRP1 was less resistant than the Cys43Ala mutant with the N1 fragment appearing at a trypsin:protein ratio of 1:250.
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ABCC1 p.Cys43Ala 12731862:136:47
status: NEW154 In contrast, E217 G uptake by membrane vesicles prepared from Cys43Ala-MRP1 expressing cells increased 40% relative to uptake by WT-MRP1.
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ABCC1 p.Cys43Ala 12731862:154:62
status: NEW173 Consequently, we examined the resistance of cells expressing mutant MRP1 molecules harboring substitutions of Cys residues in MSD1 (Cys43Ala, Cys43Ser, Cys49Ala, Cys49Ser, Cys190Ala, and Cys190Ser) andCL3(Cys208Ala,Cys208Ser,Cys265Ala,andCys265Ser), to sodium arsenite and potassium antimony tartrate.
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ABCC1 p.Cys43Ala 12731862:173:132
status: NEW181 The Cys43Ala, Cys43Ser, Cys265Ala, and Cys265Ser MRP1 mutant expressing cell lines were 6.5-, 2.6-, 7.1-, and 5.1-fold resistant to doxorubicin, respectively, levels of resistance that did not differ significantly from the ~5-fold resistance observed with cells expressing WT-MRP1 (Figure 8A).
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ABCC1 p.Cys43Ala 12731862:181:4
status: NEW182 In contrast, the cell line expressing Cys43Ser-MRP1 was only 5-fold resistant to vincristine while the cell lines expressing Cys43Ala, Cys265Ala, and Cys265Ser-MRP1 were 21-, 19-, and 13-fold resistant, respectively, levels of resistance comparable to those observed in cells expressing WT-MRP1 (15-fold resistant) (Figure 8B).
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ABCC1 p.Cys43Ala 12731862:182:125
status: NEW192 Table 2: Sensitivity of Stably Transfected HeLa Cells Expressing Wild-Type and Cys-Substituted MRP1 to Sodium Arsenite and Potassium Antimony Tartrate relative resistancea transfected HeLa cell line Na+ arsenite K+ antimony tartrate WT-MRP1 3.6 ( 1.3 (1) (n ) 6) 2.0 ( 0.5 (1) (n ) 7) C43A-MRP1 4.3 ( 0.7 (1.2) (n ) 3) 2.0 ( 0.4 (1) (n ) 3) C43S-MRP1 1.4 ( 0.5 (0.4)b (n ) 6) 2.8 ( 1.1 (1.4) (n ) 4) C49A-MRP1 4.0 ( 1.6 (1.1) (n ) 5) 2.6 ( 0.7 (1.3) (n ) 4) C49S-MRP1 3.0 ( 1.5 (0.8) (n ) 4) 3.0 ( 0.6 (1.5) (n ) 4) C190A-MRP1 3.8 ( 0.4 (1) (n ) 4) 4.0 ( 1.2 (2) (n ) 3) C190S-MRP1 2.9 ( 0.4 (0.8) (n ) 4) 2.7 ( 0.4 (1.4) (n ) 3) C208A-MRP1 3.0 ( 0.6 (0.8) (n ) 3) 2.4 ( 0.6 (1.2) (n ) 3) C208S-MRP1 4.2 ( 0.8 (1.2) (n ) 3) 3.8 ( 0.7 (1.9) (n ) 3) C265A-MRP1 2.5 ( 0.2 (0.7) (n ) 4) 2.3 ( 0.6 (0.9) (n ) 3) C265S-MRP1 10.2 ( 0.4 (2.8)b (n ) 5) 4.0 ( 1.7 (2) (n ) 3) a The resistance of stably transfected HeLa cells was determined using a tetrazolium-based cytotoxicity assay. The relative resistance factors were obtained by dividing the IC50 values for wild-type or Cys mutant MRP1 transfected cells by the IC50 values for empty vector control transfected cells and were normalized for differences in MRP1 expression levels.
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ABCC1 p.Cys43Ala 12731862:192:285
status: NEW203 The most significant decreases were observed in membranes from cells expressing the Cys43Ala, Cys49Ala, Cys148Ala, and Cys265Ala MRP1 mutants, suggesting that an alteration in the conformation of these proteins has occurred that decreases the accessibility of specific trypsin cleavage sites in CL3 (appearance of N2 fragment) and, in some cases, in the linker region of the protein between NBD1 and MSD3 (appearance of N1 fragment).
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ABCC1 p.Cys43Ala 12731862:203:84
status: NEW