ABCC1 p.Cys32Ala
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PMID: 12119019
[PubMed]
Chen Q et al: "Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1."
No.
Sentence
Comment
158
Wild-type and mutant (C7A, C32A, and C7/32A) human MRP1-transfected HEK293 cells were probed with IU15H6 (thin line), IU2H10 (thick line), and QCRL-1 (dotted line) (Centocor) in the absence (A) or presence (B) of 0.2% saponin followed by incubation with FITC-conjugated anti-mouse IgG for FACS analysis.
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ABCC1 p.Cys32Ala 12119019:158:27
status: NEW
PMID: 12235150
[PubMed]
Yang Y et al: "Structural and functional consequences of mutating cysteine residues in the amino terminus of human multidrug resistance-associated protein 1."
No.
Sentence
Comment
49
Engineering Human MRP1 Constructs-Mutations of Cys7 and Cys32 to Ala were generated using the TransformerTM site-directed mutagenesis kit (Clontech, Palo Alto, CA).
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ABCC1 p.Cys32Ala 12235150:49:56
status: NEW53 The NheI-BamHI fragments containing the wild type or desired mutations from pGEM-4Z together with a BamHI-NotI fragment from pRc/RSV-MRP were ligated into pcDNA3.1(ϩ) vector linearized with NheI and NotI to create pcDNA3.1(ϩ)-MRP1WT , pcDNA3.1(ϩ)-MRP1C7A , pcDNA3.1(ϩ)-MRP1C32A , and pcDNA3.1(ϩ)- MRP1C7A/C32A .
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ABCC1 p.Cys32Ala 12235150:53:335
status: NEW54 To generate wild type and mutant pCEP4-MRP1281N constructs, the plasmids pcDNA3.1(ϩ)-MRP1WT , -MRP1C7A , -MRP1C32A , and -MRP1C7A/C32A were digested with BamHI and blunted with Klenow followed by digestion with NheI.
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ABCC1 p.Cys32Ala 12235150:54:136
status: NEW63 For stable transfection, 5 g of pcDNA3.1(ϩ)- MRP1WT , -MRP1C7A , -MRP1C32A , or -MRP1C7A/C32A were transfected into HEK293 cells using LipofectAMINE according to the manufacturer`s instructions.
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ABCC1 p.Cys32Ala 12235150:63:103
status: NEW108 To investigate whether these two cysteine residues are functionally important, we mutated them to alanine in human MRP1 and created MRP1C7A , MRP1C32A , and MRP1C7A/C32A .
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ABCC1 p.Cys32Ala 12235150:108:165
status: NEW115 To further determine whether the mutant proteins were properly routed to the cell surface, we performed an indirect immunofluorescence staining of these cells with QCRL-1. As shown in Fig. 3, the cells expressing MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A all showed plasma membrane staining.
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ABCC1 p.Cys32Ala 12235150:115:255
status: NEW133 The calculated initial transport rates for MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A after correction for protein expression level were 35.9 Ϯ 4.9, 6.0 Ϯ 1.5, 26.5 Ϯ 1.0, and 6.9 Ϯ 1.5 pmol/mg/min, respectively.
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ABCC1 p.Cys32Ala 12235150:133:85
status: NEW134 The corrected initial [3 H]LTC4 transport rates of the mutant MRP1C7A , MRP1C32A , and MRP1C7A/C32A were about 17, 74, and 19% of that of MRP1WT , respectively.
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ABCC1 p.Cys32Ala 12235150:134:95
status: NEW136 We next measured the [3 H]LTC4 transport Km and Vmax of MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A .
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ABCC1 p.Cys32Ala 12235150:136:98
status: NEW138 However, the Vmax of MRP1C7A and MRP1C7A/C32A corrected for MRP1 expression level was 5-7-fold less than that of MRP1WT and MRP1C32A .
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ABCC1 p.Cys32Ala 12235150:138:41
status: NEW141 We next examined the drug resistance profile of transfectants expressing MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A to colchicine, doxorubicin, vincristine, and VP16 as described under "Experimental Procedures."
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ABCC1 p.Cys32Ala 12235150:141:115
status: NEW151 We thus tested the reactivity of IU2H10 to cells expressing wild type and mutant human MRP1 in comparison with the monoclonal antibody QCRL-1. As shown in Fig. 6, the IU2H10 staining profile of MRP1C7A and MRP1C7A/C32A appears to superimpose with QCRL-1 staining, whereas the IU2H10 and QCRL-1 staining traces of MRP1WT have a 10-fold difference in intensity.
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ABCC1 p.Cys32Ala 12235150:151:214
status: NEW162 A, membrane proteins (2, 1, and 0.5 g) from stable HEK293 cells transfected with vector (V), human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were subjected to SDS-PAGE and Western blot analysis probed with monoclonal antibody QCRL-1.
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ABCC1 p.Cys32Ala 12235150:162:149
status: NEW166 B, membrane proteins of human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were treated with or without Peptide N-glycosidase F and then subjected to SDS-PAGE and Western blot analysis as in A.
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ABCC1 p.Cys32Ala 12235150:166:72
status: NEW170 HEK293 cells expressing human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A grown on glass coverslips were stained with MRP1-specific monoclonal antibody QCRL-1 followed by incubation with a FITC-conjugated goat anti-mouse IgG F(abЈ)2 fragment.
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ABCC1 p.Cys32Ala 12235150:170:72
status: NEW173 However, MRP1C7A and MRP1C7A/C32A mutants generated additional peptide fragments (ϳ23 and 97 kDa) detected by MRPr1 that were not observed with MRP1WT (see the peptides indicated by arrowheads in the top panel of Fig. 7).
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ABCC1 p.Cys32Ala 12235150:173:29
status: NEW175 A relatively minute quantity of the 23- and 97-kDa peptides were also produced from MRP1C32A , suggesting that the conformation at the amino terminus of human MRP1C32A has also been changed but to a much less degree as compared with MRP1C7A and MRP1C7A/C32A .
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ABCC1 p.Cys32Ala 12235150:175:253
status: NEW181 Membrane vesicles from cells expressing human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were incubated with [3 H]LTC4 in the presence of ATP (closed symbols) or AMP (open symbols) for the time indicated.
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ABCC1 p.Cys32Ala 12235150:181:88
status: NEW183 TABLE I Kinetics parameters of LTC4 transport by vesicles from transfectants expressing MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A Km Vmax Vmax (corrected) MRP1WT 194 Ϯ 20 89 Ϯ 9 89 MRP1C7A 175 Ϯ 20 67 Ϯ 13 13 MRP1C32A 175 Ϯ 48 116 Ϯ 23 83 MRP1C7A/C32A 176 Ϯ 12 137 Ϯ 26 16 DTT at room temperature for 30 min and then subjected to SDS-PAGE and Western blot analysis.
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ABCC1 p.Cys32Ala 12235150:183:130
status: NEWX
ABCC1 p.Cys32Ala 12235150:183:288
status: NEW189 Stable HEK293 transfectants expressing human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were tested for their resistance to colchicine, doxorubicin, vincristine, and VP16 at different concentrations using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
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ABCC1 p.Cys32Ala 12235150:189:87
status: NEW194 Stable HEK293 transfectants expressing human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were probed with irrelevant antibody IU15H6 (thin solid line), MRP1-specific antibody IU2H10 (thick solid line), and QCRL-1 (dotted line) in the presence of 0.2% saponin followed by incubation with FITC-conjugated anti-mouse IgG for FACS analysis.
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ABCC1 p.Cys32Ala 12235150:194:87
status: NEW195 MRP1281N-C7A/C32A ) significantly reduced the dimer formation (Fig. 8, A, lanes 3 and 7 and B), whereas mutation of Cys32 (MRP1281N-C32A ) did not (Fig. 8, A, lane 5, and B) under nonreducing conditions.
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ABCC1 p.Cys32Ala 12235150:195:13
status: NEWX
ABCC1 p.Cys32Ala 12235150:195:132
status: NEW224 10 g of membrane proteins from cells expressing human MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A were digested with trypsin, and the membrane-associated fragments were then pelleted for SDS-PAGE and Western blot analysis using monoclonal antibody MRPr1 (top panel) and QCRL-1 (bottom panel).
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ABCC1 p.Cys32Ala 12235150:224:104
status: NEW247 Crude membranes from transiently transfected HEK293 cells expressing human MRP1281N-WT , MRP1281N-C7A , MRP1281N-C32A , and MRP1281N-C7A/C32A were treated with SDS-PAGE sample buffer in the absence or presence of 100 mM DTT before being subjected to SDS-PAGE and Western blot analysis using monoclonal antibody MRPr1 as a probe (A).
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ABCC1 p.Cys32Ala 12235150:247:113
status: NEWX
ABCC1 p.Cys32Ala 12235150:247:137
status: NEW
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
222
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.
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ABCC1 p.Cys32Ala 12731862:222:150
status: NEW
PMID: 21199866
[PubMed]
Fukuda Y et al: "Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8."
No.
Sentence
Comment
66
Using this wild-type construct as a template, we introduced a point mutation at Cys-32 to generate hMRP1-MSD0-C32A-GFP by using the QuikChange mutagenesis kit and the primers listed in the supplemental table.
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ABCC1 p.Cys32Ala 21199866:66:110
status: NEW89 Indirect Immunofluorescence Microscopy-HEK293 cells were seeded on poly-L-lysine-coated coverslips 24 h before transfection with plasmid DNA (50 ng) encoding hMRP1-MSD0-GFP or hMRP1-MSD0-C32A-GFP by using Lipofectamine Plus according to the manufacturer`s protocols.
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ABCC1 p.Cys32Ala 21199866:89:187
status: NEW278 HEK293 cells were transfected with a plasmid encoding an MRP1-MSD0-GFP chimera containing either wild-type MSD0 or MSD0 with a C32A substitution.
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ABCC1 p.Cys32Ala 21199866:278:127
status: NEW282 In contrast, the construct with the C32A substitution was Endo H-sensitive.
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ABCC1 p.Cys32Ala 21199866:282:36
status: NEW305 D, MRP1-MSD0-GFP containing either wild-type MRP1 or a C32A-MRP1 mutant was transiently expressed in HEK293 cells.
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ABCC1 p.Cys32Ala 21199866:305:55
status: NEW306 Merged indirect immunofluorescence microscopy images (left) show the wild-type protein (green) on the cell surface, whereas the C32A mutant protein remains in the ER where it co-localizes with the ER protein protein-disulfide isomerase (PDI) (red, which is shown as yellow in the co-localized cell).
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ABCC1 p.Cys32Ala 21199866:306:128
status: NEW65 Using this wild-type construct as a template, we introduced a point mutation at Cys-32 to generate hMRP1-MSD0-C32A-GFP by using the QuikChange mutagenesis kit and the primers listed in the supplemental table.
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ABCC1 p.Cys32Ala 21199866:65:110
status: NEW88 Indirect Immunofluorescence Microscopy-HEK293 cells were seeded on poly-L-lysine-coated coverslips 24 h before transfection with plasmid DNA (50 ng) encoding hMRP1-MSD0-GFP or hMRP1-MSD0-C32A-GFP by using Lipofectamine Plus according to the manufacturer`s protocols.
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ABCC1 p.Cys32Ala 21199866:88:187
status: NEW