ABCMdb

PMID: 12731862

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 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC1 p.Cys43Ser 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. Login to comment 54 ABCC1 p.Cys43Ser ABCC1 p.Cys148Ala ABCC1 p.Cys85Ser ABCC1 p.Cys265Ser ABCC1 p.Cys190Ala ABCC1 p.Cys49Ala ABCC1 p.Cys148Ser ABCC1 p.Cys265Ala ABCC1 p.Cys85Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser 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 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala 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 110 ABCC1 p.Cys85Ser ABCC1 p.Cys190Ala ABCC1 p.Cys85Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser 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)]. Login to comment 111 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser 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 112 ABCC1 p.Cys265Ser Finally, Cys265Ser expressing cells had a unique staining pattern, which was stippled and resembled vacuolar staining. Login to comment 124 ABCC1 p.Cys43Ser ABCC1 p.Cys85Ala The tryptic digest patterns of Cys43Ser and Cys85Ala-MRP1 were almost indistinguishable from that of WT-MRP1, with the appearance of the N1 fragment and faint N2 bands at trypsin:protein ratios of 1:10 000 and 1:1000, respectively. The tryptic digest patterns for vesicles prepared from mutants FIGURE 2: Expression levels of wild-type and Cys-substituted MRP1 in stably transfected HeLa cells. Login to comment 130 ABCC1 p.Cys43Ser ABCC1 p.Cys148Ala ABCC1 p.Cys85Ser ABCC1 p.Cys265Ser ABCC1 p.Cys190Ala ABCC1 p.Cys49Ala ABCC1 p.Cys148Ser ABCC1 p.Cys265Ala ABCC1 p.Cys85Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser 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 132 ABCC1 p.Cys85Ser ABCC1 p.Cys265Ser ABCC1 p.Cys190Ala ABCC1 p.Cys190Ala ABCC1 p.Cys148Ser ABCC1 p.Cys49Ser ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser ABCC1 p.Cys208Ala ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser ABCC1 p.Cys208Ser 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. Login to comment 133 ABCC1 p.Cys85Ser ABCC1 p.Cys265Ser ABCC1 p.Cys148Ser ABCC1 p.Cys190Ser For all four of these CysfSer mutants, the N1 tryptic fragment appeared at a trypsin:protein ratio of 1:1000 while the N2 fragment appeared at ratios of 1:500 for Cys148Ser and 1:250 for Cys85Ser, Cys190Ser, and Cys265Ser. Login to comment 134 ABCC1 p.Cys148Ala ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala 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 135 ABCC1 p.Cys148Ala ABCC1 p.Cys49Ala ABCC1 p.Cys43Ala 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. Login to comment 136 ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala 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 ABCC1 p.Cys265Ala 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 ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys208Ala 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 148 ABCC1 p.Cys49Ala ABCC1 p.Cys49Ser Thus, similar to Cys49Ala-MRP1, Cys49Ser-MRP1 showed a 30% reduction in relative LTC4 transport activity. Login to comment 149 ABCC1 p.Cys85Ser ABCC1 p.Cys85Ala However, in contrast to Cys85Ala-MRP1, which transported LTC4 at levels similar to WT-MRP1, Cys85Ser-MRP1 showed a 38% reduction in LTC4 uptake. Login to comment 150 ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser Similar to Cys208Ala-MRP1, Cys208Ser-MRP1 LTC4 transport activity was increased 30%. Login to comment 151 ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala In contrast, unlike Cys265Ala-MRP1, which showed increased activity, Cys265Ser-MRP1 exhibited LTC4 uptake activity similar to WT-MRP1. Login to comment 153 ABCC1 p.Cys148Ala ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys85Ala 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 154 ABCC1 p.Cys43Ala In contrast, E217 G uptake by membrane vesicles prepared from Cys43Ala-MRP1 expressing cells increased 40% relative to uptake by WT-MRP1. Login to comment 156 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys148Ser ABCC1 p.Cys49Ser Consistent with their Ala-substituted counterparts, Cys49Ser-MRP1, Cys148Ser- MRP1, and Cys265Ser-MRP1 showed moderately reduced E217 G uptake (34-50%) while uptake by Cys43Ser-MRP1 was increased by 40% relative to WT-MRP1. Login to comment 157 ABCC1 p.Cys85Ser ABCC1 p.Cys85Ala In contrast to the Cys85Ala mutant, E217 G uptake by Cys85Ser-MRP1 was similar to WT-MRP1. Login to comment 158 ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser 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%. Login to comment 160 ABCC1 p.Cys43Ser ABCC1 p.Cys208Ser Thus, membrane vesicles prepared from the Cys43Ser-MRP1 transfected cell line showed a 40% increase in GSH uptake whereas Cys208Ser-MRP1 vesicles showed a 50% decrease in GSH uptake relative to WT-MRP1. Login to comment 173 ABCC1 p.Cys43Ser ABCC1 p.Cys190Ala ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser 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 177 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser Thus, the arsenite resistance of cells expressing the TM1 Cys43Ser mutant was 2.5-fold lower than the cell line expressing WT-MRP1 whereas cells expressing Cys265Ser MRP1 were approximately 3-fold more resistant to this heavy metal oxyanion (Table 2). Login to comment 178 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser Representative chemosensitivity assays illustrating the sodium arsenite resistance of cell lines expressing the Cys43Ser and Cys265Ser MRP1 mutants are shown in Figure 7. Login to comment 179 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser The HeLa cell lines expressing Cys43Ser-MRP1 and Cys265Ser-MRP1 that showed changes in arsenite resistance were also tested for their sensitivity to the anticancer drugs vincristine and doxorubicin. Login to comment 181 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala 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 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala 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 185 ABCC1 p.Cys85Ser ABCC1 p.Cys148Ser ABCC1 p.Cys190Ser The lowest expression levels were observed with the MSD1 mutants Cys85Ser, Cys148Ser, and Cys190Ser, which were FIGURE 6: Uptake of [3H]LTC4, [3H]E217 G, and [3H]GSH by inside-out membrane vesicles prepared from wild-type and Cys-substituted MRP1 transfected HeLa cells. Login to comment 192 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys190Ala ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala ABCC1 p.Cys49Ser ABCC1 p.Cys190Ser ABCC1 p.Cys208Ala ABCC1 p.Cys208Ser 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 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser ABCC1 p.Cys265Ala 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 199 ABCC1 p.Cys43Ser The disrupted membrane trafficking of the Cys43Ser MRP1 mutant is of particular interest since a single nucleotide polymorphism resulting in this amino acid substitution has recently been reported in a Japanese population (29). Login to comment 203 ABCC1 p.Cys148Ala ABCC1 p.Cys49Ala ABCC1 p.Cys265Ala ABCC1 p.Cys43Ala 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 207 ABCC1 p.Cys43Ser In fact, one of the mutants that showed extensive intracellular staining, Cys43Ser-MRP1, had LTC4 and E217 G transport activity that was greater than or equal to that of WT-MRP1. Login to comment 208 ABCC1 p.Lys332Asp This contrasts with other amino acid substitutions of MRP1 (such as when Lys332 is replaced by Asp or Leu) that completely eliminate LTC4 transport without disrupting plasma membrane trafficking (43). Login to comment 210 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser All Cys-substituted proteins were present to a certain extent in the plasma membrane of the FIGURE 7: Resistance of HeLa cells expressing Cys43Ser and Cys265Ser mutant MRP1 to sodium arsenite. Login to comment 211 ABCC1 p.Cys265Ser HeLa cell lines stably transfected with the pcDNA3.1(-) vector (O), pcDNA3.1(-)- MRP1K (b), and (A) pcDNA3.1(-)C43S-MRP1K (9) or (B) pcDNA3.1(-)-C265S-MRP1K (9) were exposed to sodium arsenite for 72 h at 37 °C at the concentrations indicated, and then, cell viability was measured using a tetrazolium assay. The results shown are those of a typical experiment in which each data point represents the mean (SD of quadruplicate determinations. Login to comment 213 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser Data are not normalized for MRP1 expression; however, Cys265Ser-MRP1 and Cys43Ser-MRP1 were expressed at levels comparable to, and 1.5-fold higher than, WT-MRP1, respectively. Login to comment 214 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser Consequently, normalization of the data would not affect the relative resistance of Cys265Ser-MRP1 and would only further emphasize the decreased arsenite resistance of Cys43Ser-MRP1. Login to comment 222 ABCC1 p.Cys32Ala ABCC1 p.Cys7Ala Yang et al. (26) recently reported that when Cys7 and Cys32 in the extracellular NH2 terminus of MRP1 were replaced with Ala, the Cys7Ala but not the Cys32Ala mutant showed significant structural and functional changes. Login to comment 224 ABCC1 p.Cys7Ala However, in contrast to our findings with the seven Cys mutants examined here, LTC4 transport by Cys7Ala-MRP1 was reduced by more than 90%, providing indirect evidence that the structure of MRP1 was more disrupted in this mutant. Login to comment 225 ABCC1 p.Cys7Ala On the other hand, the apparent changes in Cys7Ala-MRP1 conformation resulted in little or no impairment of plasma membrane localization unlike several of the Cys-substituted mutants investigated here (26). Login to comment 237 ABCC1 p.Cys43Ser ABCC1 p.Cys265Ser In addition to their limited effects on organic anion transport function, the mutation of Cys residues in MSD1 and CL3 Cys had no effect on the ability of MRP1 to confer resistance to heavy metal-centered oxyanions with just two exceptions (Cys43Ser-MRP1 and Cys265Ser-MRP1). Login to comment 238 ABCC1 p.Cys43Ser Similarly, vincristine resistance was decreased only in the Cys43Ser-MRP1 mutant. Login to comment 247 ABCC1 p.Cys43Ser In addition, as noted above, Cys43Ser-MRP1 was not completely routed to the plasma membrane, and although this had no significant effect on organic anion transport, it might still contribute to changes in arsenite and vincristine resistance since previous studies have shown that these properties of MRP1 are not inextricably linked to each other (31). Login to comment 248 ABCC1 p.Cys43Ser ABCC1 p.Cys43Ser Finally, the reduced ability of Cys43Ser-MRP1 to confer resistance to arsenite and vincristine is of potential clinical relevance because as mentioned previously, Ito et al. (29) have reported the occurrence of a naturally occurring Cys43Ser polymorphism in healthy Japanese subjects. Login to comment 253 ABCC1 p.Cys265Ser Thus, one possible explanation for the increased arsenite resistance of Cys265Ser-MRP1 is that arsenite could be forming a complex with the mutant protein that is not held with as high affinity as WT-MRP1. Login to comment 255 ABCC1 p.Cys265Ser Another possible explanation is that substitution of Cys265 with Ser but not Ala results in a conformational change within CL3 that indirectly increases the efficiency with which arsenite interacts with the protein. Login to comment