ABCC1 p.Cys49Ala
| Predicted by SNAP2: | A: N (93%), D: D (71%), E: D (53%), F: N (82%), G: N (53%), H: D (53%), I: N (82%), K: D (59%), L: N (82%), M: N (66%), N: N (66%), P: D (63%), Q: D (53%), R: D (59%), S: N (82%), T: N (87%), V: N (93%), W: D (80%), Y: N (53%), |
| Predicted by PROVEAN: | A: N, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: N, T: N, V: N, W: N, Y: N, |
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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.Cys49Ala 12731862:54:327
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.Cys49Ala 12731862:130:298
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.Cys49Ala 12731862:134:76
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.Cys49Ala 12731862:135:142
status: NEW147 Ala substitution of Cys49 resulted in a 37% decrease in LTC4 transport activity while replacement of Cys208 and Cys265 with Ala resulted in an approximate 30% increase in activity. Four of the seven CysfSer MRP1 mutants also transported LTC4 at levels comparable to those of WT-MRP1; however, some differences between the transport activities of the Ala and Ser substituted mutants were noted (Figure 6B).
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ABCC1 p.Cys49Ala 12731862:147:0
status: NEW148 Thus, similar to Cys49Ala-MRP1, Cys49Ser-MRP1 showed a 30% reduction in relative LTC4 transport activity.
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ABCC1 p.Cys49Ala 12731862:148:17
status: NEW153 Thus, a moderate (32-42%) decrease in E217 G uptake activity was observed for four of the five mutants (Cys49Ala-MRP1, Cys85Ala-MRP1, Cys148Ala-MRP1, and Cys265Ala-MRP1).
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ABCC1 p.Cys49Ala 12731862:153:104
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.Cys49Ala 12731862:173:152
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.Cys49Ala 12731862:192:400
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.Cys49Ala 12731862:203:94
status: NEW