ABCMdb

PMID: 12235150

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 [PubMed]

Sentences

No. Mutations Sentence Comment

49 ABCC1 p.Cys32Ala ABCC1 p.Cys7Ala Engineering Human MRP1 Constructs-Mutations of Cys7 and Cys32 to Ala were generated using the TransformerTM site-directed mutagenesis kit (Clontech, Palo Alto, CA). Login to comment 53 ABCC1 p.Cys32Ala 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 . Login to comment 54 ABCC1 p.Cys32Ala 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. Login to comment 63 ABCC1 p.Cys32Ala 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. Login to comment 108 ABCC1 p.Cys32Ala 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 . Login to comment 115 ABCC1 p.Cys32Ala 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. Login to comment 133 ABCC1 p.Cys32Ala 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. Login to comment 134 ABCC1 p.Cys32Ala 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. Login to comment 136 ABCC1 p.Cys32Ala We next measured the [3 H]LTC4 transport Km and Vmax of MRP1WT , MRP1C7A , MRP1C32A , and MRP1C7A/C32A . Login to comment 138 ABCC1 p.Cys32Ala However, the Vmax of MRP1C7A and MRP1C7A/C32A corrected for MRP1 expression level was 5-7-fold less than that of MRP1WT and MRP1C32A . Login to comment 141 ABCC1 p.Cys32Ala 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." Login to comment 151 ABCC1 p.Cys32Ala 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. Login to comment 162 ABCC1 p.Cys32Ala 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. Login to comment 166 ABCC1 p.Cys32Ala 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. Login to comment 170 ABCC1 p.Cys32Ala 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. Login to comment 173 ABCC1 p.Cys32Ala 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). Login to comment 175 ABCC1 p.Cys32Ala 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 . Login to comment 181 ABCC1 p.Cys32Ala 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. Login to comment 183 ABCC1 p.Cys32Ala ABCC1 p.Cys32Ala 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. Login to comment 189 ABCC1 p.Cys32Ala 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. Login to comment 194 ABCC1 p.Cys32Ala 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. Login to comment 195 ABCC1 p.Cys32Ala ABCC1 p.Cys32Ala 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. Login to comment 224 ABCC1 p.Cys32Ala 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). Login to comment 247 ABCC1 p.Cys32Ala ABCC1 p.Cys32Ala 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). Login to comment