ABCC1 p.Cys208Ala
| Predicted by SNAP2: | A: N (53%), D: D (80%), E: D (85%), F: D (63%), G: D (71%), H: D (59%), I: D (63%), K: D (75%), L: D (66%), M: D (75%), N: N (53%), P: D (80%), Q: D (66%), R: D (71%), S: N (53%), T: D (63%), V: D (63%), W: D (85%), Y: D (59%), |
| 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
110 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)].
X
ABCC1 p.Cys208Ala 12731862:110:189
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.
X
ABCC1 p.Cys208Ala 12731862:130:490
status: NEW132 Cys49Ser, Cys190Ala, Cys208Ala, and Cys208Ser also closely matched those of WT-MRP1 except that the N1 fragment did not appear until a trypsin:protein ratio of 1:1000 for Cys190Ala-MRP1 and the N2 fragments of Cys49Ser-MRP1, Cys208Ala-MRP1, and Cys208Ser-MRP1 did not appear until trypsin:protein ratios of 1:500, 1:250, and 1:500, respectively. The remaining CysfSer MRP1 mutants (Cys85Ser, Cys148Ser, Cys190Ser, and Cys265Ser) were more resistant to trypsinolysis than WT-MRP1 but less resistant than their respective Ala partners.
X
ABCC1 p.Cys208Ala 12731862:132:21
status: NEWX
ABCC1 p.Cys208Ala 12731862:132:225
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).
X
ABCC1 p.Cys208Ala 12731862:147:101
status: NEW150 Similar to Cys208Ala-MRP1, Cys208Ser-MRP1 LTC4 transport activity was increased 30%.
X
ABCC1 p.Cys208Ala 12731862:150:11
status: NEW158 In addition, unlike Cys208Ala-MRP1, which transported E217 G at a level similar to WT-MRP1, E217 G uptake by Cys208Ser-MRP1 was reduced by 35%.
X
ABCC1 p.Cys208Ala 12731862:158:20
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.
X
ABCC1 p.Cys208Ala 12731862:173:205
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.
X
ABCC1 p.Cys208Ala 12731862:192:630
status: NEW[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.
Comments [show]
None has been submitted yet.
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.
X
ABCC1 p.Cys208Ala 22511347:64:97
status: NEWX
ABCC1 p.Cys208Ala 22511347:64:103
status: NEW191 The activity of ⌬MRP1204-282cysless containing the C208A and C265A mutations was most dramatically affected and was decreased by ϳ70% (Fig. 5B, top panel).
X
ABCC1 p.Cys208Ala 22511347:191:58
status: NEW192 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).
X
ABCC1 p.Cys208Ala 22511347:192:59
status: NEW190 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).
X
ABCC1 p.Cys208Ala 22511347:190:57
status: NEW