ABCMdb

ABCC1 p.Cys265Ala

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Publications

PMID: 19200005 [PubMed] Porcelli L et al: "Intracellular trafficking of MDR transporters and relevance of SNPs."

No. Sentence Comment

241 In particular, the cell lines expressing the variants Cys43Ser-ABCC1, Cys 265Ala-ABCC1 and Cys265Ser-ABCC1 exhibited severely disrupted plasma membrane trafficking [116]. Login to comment

PMID: 12731862 [PubMed] Leslie EM et al: "Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)."

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). Login to comment
109 Thus, the cell lines expressing the TM1 mutant Cys43Ser-MRP1 (Figure 3D) and the CL3 mutants Cys265Ala-MRP1 (Figure 3O) and Cys265Ser-MRP1 (Figure 3P) exhibited severely disrupted plasma membrane trafficking. Login to comment
111 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. Login to comment
130 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. Login to comment
134 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. Login to comment
136 The Cys265Ala MRP1 was less resistant than the Cys43Ala mutant with the N1 fragment appearing at a trypsin:protein ratio of 1:250. Login to comment
137 The cleavage of the N1 fragment into the smaller N2 and N3 fragments was most affected by the substitution of Cys265 with Ala since the N2 fragment of this mutant was not detected until a trypsin: protein ratio of 1:10. Login to comment
147 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). Login to comment
151 In contrast, unlike Cys265Ala-MRP1, which showed increased activity, Cys265Ser-MRP1 exhibited LTC4 uptake activity similar to WT-MRP1. Login to comment
153 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). Login to comment
173 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. Login to comment
181 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). Login to comment
182 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). Login to comment
192 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. Login to comment
197 In addition, several Cys mutants did not appear fully routed to the plasma membrane, with the most severely disrupted pattern of subcellular localization being observed with the TM1 mutant Cys43Ser and the CL3 mutants Cys265Ala and Cys265Ser. Login to comment
203 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). Login to comment

PMID: 22511347 [PubMed] Qin L et al: "Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function."

No. Sentence Comment

64 Name Inserted (ϩ) or Deleted (-) Restriction Enzymes Primer Sequence (5Ј-3Јa 1 C208A N.A. CCCTAATCCCGCCCCAGAGTCCAG 2 C265A -BsmI GGAAGAAGGAAGCCGCCAAGACTAG 3 C375A -PstI GTCACTGCCGCCTTGCAGACCCTCG 4 C388A N.A. CTTCCACATCGCCTTCGTCAGTGG 5 C555A ϩSpoI/NruI CACCTGGGTCGCGACGCCCTTTCTG 6 C563A ϩBalI/MscI GGTGGCCTTGGCCACATTTGCCGTC 7 C682A ϩNarI CCAGGTGGGCGCCGGAAAGTCGTC 8 C730A ϩEspI, SacI ATCCTTTTTGGAGCTCAGCTGGAGG 9 C744A -StuI GATACAGGCCGCTGCCCTCCTCC 10 C984A ϩBalI/MscI TTCCTTTTCATGGCCAACCATGTGTCC 11 C1047A ϩSacI/SstI TTGGCTTCCCGAGCTCTGCACGTGG 12 C1105A ϩPvuI CATTGGTGCCGCGATCGTTATCCTG 13 C1205A N.A. GCGGCTGGAGGCTGTGGGCAACTG 14 C1209A ϩPvuI GTGTGGGCAACGCGATCGTTCTGTTTG 15 C1299A ϩMluI TTCCGGAACTACGCGTTGCGCTACCGAG 16 C1423A ϩPstI CTAGACCATGAAGCTGCAGAAGGC 17 C1439A ϩPflMI CCAGCTTGTGGCCCTAGCCCGGG 18 C1479A N.A. GTTCGAGGACGCCACCGTCCTCAC 19 Rec L N.A. GAAACCATCCACGACCCTAATCCCGCCCCAGAG 20 Rec R N.A. GGATTAGGGTCGTGGATGGTTTCCGAGAACAG 21 KbnL N.A. CATGGTACCATGGCGCTCCGGGGCTTCTGCAGC 22 KbnR N.A. GGCAGGATCCTTGGAGGAGTACACAACCTTC N.A., not applicable. Login to comment
191 The activity of ⌬MRP1204-282cysless containing the C208A and C265A mutations was most dramatically affected and was decreased by ϳ70% (Fig. 5B, top panel). Login to comment
192 Subsequently, we found that LTC4 transport activity of the C208A and C265A double mutant could be substantially recovered by growth of the HEK transfectants at 28°C (Fig. 5B, bottom panel). Login to comment
190 The activity of èc;MRP1204-282cysless containing the C208A and C265A mutations was most dramatically affected and was decreased by b03;70% (Fig. 5B, top panel). Login to comment