ABCMdb

ABCC1 p.Cys265Ala

Predicted by SNAP2:	A: N (78%), D: N (53%), E: N (61%), F: N (78%), G: N (57%), H: N (87%), I: N (78%), K: N (72%), L: N (87%), M: N (82%), N: N (78%), P: N (53%), Q: N (82%), R: N (72%), S: N (78%), T: N (82%), V: N (87%), W: N (72%), Y: N (87%),
Predicted by PROVEAN:	A: N, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, V: N, W: D, Y: D,

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[hide] Intracellular trafficking of MDR transporters and ... Curr Top Med Chem. 2009;9(2):197-208. Porcelli L, Lemos C, Peters GJ, Paradiso A, Azzariti A
Intracellular trafficking of MDR transporters and relevance of SNPs.
Curr Top Med Chem. 2009;9(2):197-208., [PMID:19200005]

Abstract [show]
Multi-drug resistance (MDR) frequently contributes to the failure of chemotherapeutic treatments in cancer patients. Mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Among them, the ATP-Binding Cassette (ABC) family of proteins plays a pivotal role. Processes of cellular distribution and subcellular localization of MDR-ABC proteins are not yet well explored and to enlighten these topics could be crucial to understand cellular drug uptake and retention. In this review, we analysed literature data concerning i) intracellular trafficking of MDR-ABC proteins (BCRP, P-gp and MRP1) and ii) mechanisms altering their cellular localization and trafficking. Moreover, we describe single nucleotide polymorphisms (SNP) that have been reported for some multidrug resistance (MDR) transporters, such as BCRP and P-gp, emphasizing their ability to affect the expression, function and localization of the transporters, with implications on drug resistance phenotypes.

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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

[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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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

[hide] Effect of multiple cysteine substitutions on the f... Drug Metab Dispos. 2012 Jul;40(7):1403-13. Epub 2012 Apr 16. Qin L, Tam SP, Deeley RG
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.
Drug Metab Dispos. 2012 Jul;40(7):1403-13. Epub 2012 Apr 16., [PMID:22511347]

Abstract [show]
Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (DeltaMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) DeltaMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of DeltaMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of DeltaMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17beta-estradiol 17-beta-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

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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