ABCC1 p.Arg1202Gly
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PMID: 12721111
[PubMed]
Ren XQ et al: "Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1."
No.
Sentence
Comment
7
Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C4 (LTC4) transport activity and conferred Vincristine resistance in LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:7:42
status: NEW53 MRP1 constructs encoding R1202G was generated in PCR reaction using the forward primers: 50 -GGGCTCGAGTGT GTGGGCAACTGC-30 (bold denotes a mismatched base encoding the mutation).
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ABCC1 p.Arg1202Gly 12721111:53:25
status: NEW116 To investigate the role of the charged amino acid in the azido AG-A photolabelling of MRP1, we replaced the Arg1202 with Gly (R1202G).
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ABCC1 p.Arg1202Gly 12721111:116:108
status: NEWX
ABCC1 p.Arg1202Gly 12721111:116:126
status: NEW118 Figure 4a shows that the expression level of R1202G mutant was comparable to that of wild-type MRP1.
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ABCC1 p.Arg1202Gly 12721111:118:45
status: NEW119 The membrane vesicles expressing the wild-type and R1202G MRP1s were analyzed for their abilities to interact with azido AG-A in a GSH-dependent manner.
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ABCC1 p.Arg1202Gly 12721111:119:51
status: NEW120 As shown in Figure 4b, wild-type MRP1 was photolabelled with azido AG-A in a GSH-dependent manner; however, the GSH-dependent photolabelling of MRP1 R1202G with [125 I]azido AG-A was greatly reduced (Figure 4b).
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ABCC1 p.Arg1202Gly 12721111:120:149
status: NEW121 To test whether the mutation affects the function of MRP1, the LTC4 transport activity of MRP1 R1202G was examined.
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ABCC1 p.Arg1202Gly 12721111:121:95
status: NEW122 As shown in Figure 5a, MRP1 R1202G efficiently transported LTC4.
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ABCC1 p.Arg1202Gly 12721111:122:28
status: NEW123 The apparent Km values of MRP1 R1202G and wild-type MRP1 for LTC4 were 147 and 117 nM, respectively (Figure 5b).
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ABCC1 p.Arg1202Gly 12721111:123:31
status: NEW124 In order to test the ability of R1202G to confer drug resistance, the wild-type MRP1 and the MRP1 R1202G mutant cDNAs were cloned into the mammalian expression vector pCIneo and transfected into LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:124:32
status: NEWX
ABCC1 p.Arg1202Gly 12721111:124:98
status: NEW139 Transfection of wild-type MRP1, or the MRP1 R1202G mutant, but not the pCIneo empty vector, resulted in drug-resistant colonies within 2 weeks.
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ABCC1 p.Arg1202Gly 12721111:139:44
status: NEW140 The drug resistance of stably transfected clones that express MRP1 or MRP1 R1202G (Figure 6a) was further tested in a cytotoxicity assay.
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ABCC1 p.Arg1202Gly 12721111:140:75
status: NEW141 As shown in Figure 6b, MRP1 and MRP1 R1202G conferred VCR resistance on LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:141:37
status: NEW142 MRP1 R1202G mutant protein was localized to the plasma membrane in a similar manner to the wild-type MRP1, indicating that the R1202G mutation did not affect the trafficking of the mutant protein (Figure 6c).
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ABCC1 p.Arg1202Gly 12721111:142:5
status: NEWX
ABCC1 p.Arg1202Gly 12721111:142:127
status: NEW143 The fact that the photolabelling of MRP1 with azido AG-A was impaired by the R1202G mutation, but the transport activity and the ability to confer resistance to VCR were retained in this mutant suggests that the R1202G mutation affected only the photolabelling, but not the binding of azido AG-A to MRP1.
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ABCC1 p.Arg1202Gly 12721111:143:77
status: NEWX
ABCC1 p.Arg1202Gly 12721111:143:212
status: NEW144 To investigate whether the MRP1 R1202G mutant can interact with AG-A, the effect of AG-A on [3 H]LTC4 uptake by wild-type MRP1 and the MRP1 R1202G mutant expressed in Sf21 insect cells was examined.
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ABCC1 p.Arg1202Gly 12721111:144:32
status: NEWX
ABCC1 p.Arg1202Gly 12721111:144:140
status: NEW145 In the presence of 2 mM GSH, AG-A inhibited uptake of [3 H]LTC4 in membrane vesicles that express MRP1 R1202G in a dose-dependent manner and the extent of the inhibition was similar to that in membrane vesicles that express wild-type MRP1 (Figure 7).
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ABCC1 p.Arg1202Gly 12721111:145:103
status: NEW146 These findings suggest that MRP1 R1202G as well as wild-type MRP1 can interact with AG-A, and Arg1202 is not critical to the binding of AG-A to MRP1, although it affects the photolabelling of azido AG-A.
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ABCC1 p.Arg1202Gly 12721111:146:33
status: NEW177 When the membrane vesicles expressing the mutant protein were photolabelled and subsequently digested with trypsin or V8 Figure 4 Effect of R1202G mutation on the GSH-dependent photolabelling of MRP1 with [125 I]azido AG-A.
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ABCC1 p.Arg1202Gly 12721111:177:140
status: NEW178 (a) Wild-type MRP1 and MRP1 R1202G were expressed in insect cells.
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ABCC1 p.Arg1202Gly 12721111:178:28
status: NEW180 (b) Wild-type MRP1 and MRP1 R1202G membrane vesicles (100 mg of membrane proteins) were photolabelled with [125 I]azido AG-A in the absence or presence of the indicated concentrations of GSH as described in the legend to Figure 2a.
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ABCC1 p.Arg1202Gly 12721111:180:28
status: NEW182 Figure 5 Effect of R1202G mutation on ATP-dependent [3 H]LTC4 transport.
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ABCC1 p.Arg1202Gly 12721111:182:19
status: NEW183 (a) Time course of ATP-dependent transport of [3 H]LTC4 by wild-type MRP1 (square) and MRP1 R1202G (triangle) mutant.
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ABCC1 p.Arg1202Gly 12721111:183:92
status: NEW184 Membrane vesicles (50 mg) expressing wild-type MRP1 or MRP1 R1202G were incubated with 1.37 nM [3 H]LTC4 at 371C in 50 ml transport buffer as described in the legend to Figure 1b in the presence or absence of 4 mM ATP at the indicated periods.
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ABCC1 p.Arg1202Gly 12721111:184:60
status: NEW187 (b) Determination of the Km values of MRP1 wild type (square) and MRP1 R1202G (triangle) for [3 H]LTC4.
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ABCC1 p.Arg1202Gly 12721111:187:71
status: NEW205 On the other hand, if one of the trypsin digestion sites is located in the cytoplasmic portion Figure 6 Effect of R1202G mutation on drug resistance in LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:205:114
status: NEW206 (a) Expression of wild-type MRP1 and the MRP1 R1202G mutant in LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:206:46
status: NEW207 Crude membranes (50 mg of protein) prepared from LLC-PK1 cells transfected with either expression vectors encoding wild-type MRP1, MRP1 R1202G, or a control empty vector (pCIneo) were analyzed on 7.5% SDS - PAGE.
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ABCC1 p.Arg1202Gly 12721111:207:136
status: NEW210 LLC-PK1 cells expressing either wild-type MRP1 (square), MRP1 R1202G (triangle), or transfected with an empty vector (rhombus) were exposed to the indicated concentrations of VCR and the survival rate was determined by MTT assay as described under Materials and methods.
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ABCC1 p.Arg1202Gly 12721111:210:62
status: NEW213 Indirect immunofluorescent staining of wild-type MRP1 and MRP1 R1202G expressed in LLC-PK1 cells was carried out with the MRPm6 mAb and a FITC-conjugated secondary antibody.
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ABCC1 p.Arg1202Gly 12721111:213:63
status: NEW215 Both wild-type MRP1 and MRP1 R1202G were localized in the plasma membrane of LLC-PK1 cells.
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ABCC1 p.Arg1202Gly 12721111:215:29
status: NEW217 Figure 7 Effect of AG-A on [3 H]LTC4 transport by wild-type MRP1 and MRP1 R1202G.
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ABCC1 p.Arg1202Gly 12721111:217:74
status: NEW218 ATP-dependent [3 H]LTC4 uptake into MRP1 and MRP1 R1202G membrane vesicles from Sf21 insect cells was measured in the presence of 2 mM GSH and 10, 30 or 100 mM of AG-A as indicated.
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ABCC1 p.Arg1202Gly 12721111:218:50
status: NEW233 Therefore, we replaced the arginine at 1202 that is proximate to the N-terminus of TM16 of MRP1 with Gly (R1202G) to investigate the role of the charged amino acid in the region where GSH-dependent azido AG-A photolabelling site resides.
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ABCC1 p.Arg1202Gly 12721111:233:106
status: NEW234 GSH-dependent photolabelling of the mutant MRP1 R1202G with [125 I]azido AG-A was considerably lower than that of wild-type MRP1.
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ABCC1 p.Arg1202Gly 12721111:234:48
status: NEW235 The impaired GSH-dependent photolabelling of MRP1 R1202G may be attributable to the impaired binding of MRP1 to AG-A, and thus the GSH-dependent photolabelling with azido AG-A was lowered.
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ABCC1 p.Arg1202Gly 12721111:235:50
status: NEW241 Alternatively, the azido AG-A photolabelling site may reside in the region nearby Arg1202 that is proximate to the N-terminus of TM16, and the replacement of Arg1202 with Gly may affect the photolabelling of this site.
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ABCC1 p.Arg1202Gly 12721111:241:158
status: NEW
PMID: 14754409
[PubMed]
Karwatsky JM et al: "Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling."
No.
Sentence
Comment
199
PAL of MRP1 R1202G was greatly reduced, although it retained transport activity and conferred vincristine resistance.
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ABCC1 p.Arg1202Gly 14754409:199:12
status: NEW
PMID: 14965249
[PubMed]
Haimeur A et al: "The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation."
No.
Sentence
Comment
276
Additional mutations of charged residues that lead to defective MRP1 transport activity (or altered drug interactions) have been described (e.g. TM16 Arg1202Gly and TM17 Arg1249Ala) but because only one or two substrates were tested in these studies, it is not possible to know whether the altered activity is relatively substrate selective or more global in nature [167, 168].
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ABCC1 p.Arg1202Gly 14965249:276:150
status: NEW
PMID: 15208328
[PubMed]
Situ D et al: "Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter."
No.
Sentence
Comment
4
In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1.
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ABCC1 p.Arg1202Gly 15208328:4:59
status: NEW116 MRP1 expression levels in membrane vesicle proteins R1202G and R1202L prepared from cells expressing the neutrally substituted Arg1202 mutants were determined as described in A.
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ABCC1 p.Arg1202Gly 15208328:116:52
status: NEW118 Immunoblots of membrane vesicle proteins prepared from cells expressing the Glu1204 mutants E1204L and E1204D were carried out as described in A. TABLE I Summary of organic anion transport activity of MRP1 mutants with substitutions of ionizable amino acids in and proximal to TM13 to TM17 of MSD3 Mutation % Wild-type MRP1 transport activitya E217betaG LTC4 E1SO4 MTX GSH TM13 R1046D 115 70 80 120 NDb TM14 D1084R Ͻ10 Ͻ10 15 25 Ͻ10 D1084E 80 20 65 90 20 TM15 R1131E 70 50 80 60 ND TM16 R1197E Ͻ10 Ͻ10 Ͻ15 Ͻ10 ND R1197K 20 Ͻ25 Ͻ20 Ͻ10 ND R1202G 115 115 75 70 ND R1202L 115 120 50 110 ND E1204L Ͻ10 50 10 110 Ͻ25 E1204D 100 115 100 115 Ͻ25 TM17 R1249D Ͻ10 Ͻ15 Ͻ10 Ͻ10 ND R1249K Ͻ10 10 Ͻ15 Ͻ10 ND a The values shown are means of duplicate or triplicate determinations and are derived from Fig. 2, 4, and 5 (see figure legends for details).
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ABCC1 p.Arg1202Gly 15208328:118:598
status: NEW124 Immunoblots showed that expression levels of the R1202G and R1202L mutants (Fig. 3B) and the E1204L and E1204D mutants (Fig. 3C) ranged from 80 to 225% of wild-type MRP1.
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ABCC1 p.Arg1202Gly 15208328:124:49
status: NEW127 At 1 min, E217betaG and LTC4 uptake by the R1202G and R1202L mutants was comparable with wild-type levels (Fig. 4, A and B).
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ABCC1 p.Arg1202Gly 15208328:127:43
status: NEW128 Levels of E1SO4 uptake by the R1202G mutant were reduced by ϳ25%, whereas uptake by the R1202L mutant was reduced by ϳ50% (Fig. 4C).
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ABCC1 p.Arg1202Gly 15208328:128:30
status: NEW129 Uptake levels of MTX by the R1202G mutant was moderately reduced (by ϳ30%), whereas uptake by R1202L was comparable with wild-type MRP1 (Fig. 4D).
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ABCC1 p.Arg1202Gly 15208328:129:28
status: NEW169 A-D, uptake of 3 H-labeled organic anions by the membrane vesicles shown in Fig. 3B which were prepared from cells transfected with empty pcDNA3.1 vector (open bars), and vector containing wild-type MRP1 cDNA (black bars), and the neutrally substituted Arg1202 mutant R1202G and R1202L cDNAs (gray bars).
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ABCC1 p.Arg1202Gly 15208328:169:268
status: NEW201 In addition to being readily expressed, the neutrally substituted mutants of TM16 Arg1202 (R1202G and R1202L) exhibited transport activities that were, in the case of most substrates, similar to those of wild-type MRP1.
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ABCC1 p.Arg1202Gly 15208328:201:91
status: NEW202 Our findings are consistent with a recent report that a neutrally substituted Arg1202 mutant (R1202G) could still transport LTC4 (28).
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ABCC1 p.Arg1202Gly 15208328:202:94
status: NEW