ABCC1 p.Cys32Ala
| Predicted by SNAP2: | A: D (53%), D: D (85%), E: D (85%), F: D (80%), G: D (66%), H: D (80%), I: D (71%), K: D (85%), L: D (66%), M: D (75%), N: D (75%), P: D (85%), Q: D (80%), R: D (85%), S: D (63%), T: D (71%), V: D (66%), W: D (85%), Y: D (80%), |
| 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, |
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[hide] Cytoplasmic retraction of the amino terminus of hu... Biochemistry. 2002 Jul 23;41(29):9052-62. Chen Q, Yang Y, Liu Y, Han B, Zhang JT
Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1.
Biochemistry. 2002 Jul 23;41(29):9052-62., 2002-07-23 [PMID:12119019]
Abstract [show]
Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.
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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[hide] Structural and functional consequences of mutating... J Biol Chem. 2002 Nov 15;277(46):44268-77. Epub 2002 Sep 13. Yang Y, Chen Q, Zhang JT
Structural and functional consequences of mutating cysteine residues in the amino terminus of human multidrug resistance-associated protein 1.
J Biol Chem. 2002 Nov 15;277(46):44268-77. Epub 2002 Sep 13., 2002-11-15 [PMID:12235150]
Abstract [show]
Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.
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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[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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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[hide] Conserved intramolecular disulfide bond is critica... J Biol Chem. 2011 Mar 11;286(10):8481-92. Epub 2011 Jan 3. Fukuda Y, Aguilar-Bryan L, Vaxillaire M, Dechaume A, Wang Y, Dean M, Moitra K, Bryan J, Schuetz JD
Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8.
J Biol Chem. 2011 Mar 11;286(10):8481-92. Epub 2011 Jan 3., [PMID:21199866]
Abstract [show]
The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.
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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