ABCB1 p.Asp555Asn

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PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."
No. Sentence Comment
165 But Hrycyna et al. [9] reported that the mutations D555N and D1200N (D555 and D1200 are presumably involved in Mg2+ binding) resulted in impaired nucleotide photoaffinity labeling only for D555N.
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ABCB1 p.Asp555Asn 16442101:165:51
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ABCB1 p.Asp555Asn 16442101:165:189
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PMID: 10529234 [PubMed] Hrycyna CA et al: "Both ATP sites of human P-glycoprotein are essential but not symmetric."
No. Sentence Comment
60 Two internal complementary primers were used, each containing the mutation of interest (G f A at position 1663 for D555N).
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ABCB1 p.Asp555Asn 10529234:60:115
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61 The coding sequence for the D555N mutant primer was 5'- CTCCTGCTGAATGAGGCCAC-3'.
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ABCB1 p.Asp555Asn 10529234:61:28
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70 The resulting plasmid was called pTM1-MDR1-D555N.
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ABCB1 p.Asp555Asn 10529234:70:43
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79 The D555N/D1200N double mutant was constructed exactly like pTM1-MDR1-D1200N except that pTM1-MDR1-D555N was used as the template for the first PCR reaction.
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ABCB1 p.Asp555Asn 10529234:79:4
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ABCB1 p.Asp555Asn 10529234:79:99
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80 The resulting expression plasmid was named pTM1-MDR1-D555N/ D1200N.
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ABCB1 p.Asp555Asn 10529234:80:53
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106 Recombinant vaccinia viruses vvT7MDR1(WT) (wild-type P-gp), vvMDR1-CM (P-gp-D1200N), vvMDR1-Mg5 (P-gp-D555N), and vvMDR1-DM (P-gp-D555N/D1200N) were constructed as previously described (28) from the corresponding pTM1-MDR1 vectors described above.
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ABCB1 p.Asp555Asn 10529234:106:102
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ABCB1 p.Asp555Asn 10529234:106:130
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163 The two mutants, designated P-gp-D555N and P-gp-D1200N, were constructed by site-directed mutagenesis, cloned into pTM1-MDR1, and transiently expressed in HeLa cells coinfected with the vaccinia virus vTF7-3 as described under Experimental Procedures.
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ABCB1 p.Asp555Asn 10529234:163:33
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165 Total extracts from HeLa cells expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, and the double mutant P-gp-D555N/ D1200N were subjected to immunoblot analysis with the monoclonal antibody C219 (32).
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ABCB1 p.Asp555Asn 10529234:165:63
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ABCB1 p.Asp555Asn 10529234:165:110
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166 While P-gp-D1200N was found to be expressed at a level comparable to wild-type P-gp, in this preparation of membranes it appears that less P-gp-D555N and P-gp D555N/D1200N were expressed compared to wild-type.
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ABCB1 p.Asp555Asn 10529234:166:144
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ABCB1 p.Asp555Asn 10529234:166:159
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169 Fluorescent substrate accumulation assays were performed on intact HeLa cells expressing wild-type P-gp, P-gp-D555N, and P-gp-D1200N with the P-gp substrate calcein-AM.
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ABCB1 p.Asp555Asn 10529234:169:110
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170 HeLa cells were transiently infected with vTF7-3 and transfected with pTM1-MDR1 (wild-type), pTM1-MDR1-D555N, and pTM1-MDR1-D1200N, with pTM1 as the negative control, and allowed to incubate for approximately 24 h. After incubation with 0.5 µM calcein-AM for 10 min, cells expressing wild-type P-gp accumulated less substrate than the pTM1 control as measured by a decrease in fluorescence intensity (Figure 2).
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ABCB1 p.Asp555Asn 10529234:170:103
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172 Strikingly, the two Walker B mutants P-gp-D555N and P-gp-D1200N were completely devoid of transport function (Figure 2).
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ABCB1 p.Asp555Asn 10529234:172:42
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178 The ability of Walker B mutants P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N to hydrolyze ATP either in the absence (basal) or presence (drug-stimulated) of 25 µM verapamil was measured as the vanadate-sensitive release of inorganic phosphate from Mg2+ ‚ATP as described under Experimental Procedures, with membrane preparations from vTF7-3-infected HeLa cells alone or vTF7-3-infected cells that were coinfected with recombinant vaccinia viruses encoding wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N.
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ABCB1 p.Asp555Asn 10529234:178:37
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ABCB1 p.Asp555Asn 10529234:178:66
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ABCB1 p.Asp555Asn 10529234:178:491
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ABCB1 p.Asp555Asn 10529234:178:520
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184 (A) vTF7-3-infected-transfected HeLa cells (500 000) expressing wild-type P-gp (bold line), P-gp-D555N (---), and P-gp-D1200N (-‚-) were subjected to FACS analysis after incubation with 5 µg of MRK-16 for 30 min at 37 °C, washing (200g, 5 min), and staining with FITC-conjugated anti-mouse IgG secondary antibody (1 µg) for 30 min at 37 °C.
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ABCB1 p.Asp555Asn 10529234:184:97
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188 HeLa cells were infected with vTF7-3 recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D12000N and harvested after 48 h. Membranes (1 µg/lane) prepared from these cells were subjected to SDS-PAGE and immunoblotting with monoclonal antibody C219 (1:1500).
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ABCB1 p.Asp555Asn 10529234:188:98
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ABCB1 p.Asp555Asn 10529234:188:127
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191 HeLa cells were infected with vTF7-3 and transfected with pTM1-MDR1 (wild type), pTM1-MDR1-D555N, and pTM1-MDR1-D1200N for 24 h. Calcein (0.5 µM) accumulation was determined in these cells by FACS after a 10 min incubation at 37 °C in the presence (thin line) and absence (bold line) of 2 µM cyclosporin A. vTF7-3-infected cells transfected with pTM1 vector DNA were included as a negative control.
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ABCB1 p.Asp555Asn 10529234:191:91
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195 Crude membrane preparations derived from HeLa cells infected with vTF7-3 alone and from cells coinfected with recombinant vaccinia viruses encoding wild-type P-gp, P-gp-D555N, P-gp-D1200N, and P-gp-D555N/D1200N were labeled with [125I]IAAP at a concentration of 7.5 nM, immunoprecipitated with anti-P-gp polyclonal antibody PEPG-2 (33), and subjected to SDS-PAGE and autoradiography (Figure 4A).
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ABCB1 p.Asp555Asn 10529234:195:169
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ABCB1 p.Asp555Asn 10529234:195:198
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202 To further demonstrate that ATP hydrolysis is essential and to evaluate the roles of the two NBDs for inhibition of [125I]IAAP labeling as a result of vanadate trapping, we analyzed the effects of the Walker B consensus motif mutations D555N, D1200N, and D555N/D1200N.
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ABCB1 p.Asp555Asn 10529234:202:236
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ABCB1 p.Asp555Asn 10529234:202:255
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207 Human P-gp-D555N and P-gp-D1200N Differ Dramatically in Their Ability To Be Labeled with [R-32P]-8-Azido-ATP.
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ABCB1 p.Asp555Asn 10529234:207:11
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210 Membrane preparations from HeLa cells infected with vTF7-3 and coinfected with vvT7MDR1 (WT) (wild-type P-gp), vvMDR1-CM (P-gp-D1200N), vvMDR1-Mg5 (P-gp-D555N), or vvMDR1-DM (P-gp-D555N/D1200N).
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ABCB1 p.Asp555Asn 10529234:210:153
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ABCB1 p.Asp555Asn 10529234:210:180
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216 (A) Wild type and the mutant P-gps D555N, D1200N, and D555N/1200N were expressed in HeLa cells by use of recombinant vaccinia viruses, and crude membranes (45 µg) were used for [125I]IAAP photoaffinity labeling.
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ABCB1 p.Asp555Asn 10529234:216:35
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ABCB1 p.Asp555Asn 10529234:216:54
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220 Wild-type (b) and mutant Pgps, D555N ([), D1200N (4), and D555N/D1200N (O) were expressed in HeLa cells by use of recombinant vaccinia viruses and crude membranes (15 µg) were used for [125I]IAAP labeling in the presence of varying concentrations of vanadate, 5 mM MgCl2, and 2.5 mM ATP.
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ABCB1 p.Asp555Asn 10529234:220:31
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ABCB1 p.Asp555Asn 10529234:220:58
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227 Strikingly, the [R-32P]-8-azido-ATP labeling observed in membranes expressing P-gp-D555N and P-gp-D555N/D1200N was severely impaired.
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ABCB1 p.Asp555Asn 10529234:227:83
status: NEW
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ABCB1 p.Asp555Asn 10529234:227:98
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229 These results suggest that the ability to bind ATP to the N-half ATP site, as determined by photoaffinity labeling, is eliminated or reduced significantly in the protein containing a mutation in the magnesium binding site (D555N).
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ABCB1 p.Asp555Asn 10529234:229:223
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232 The same experiment was subsequently performed at a saturating concentration of 77.5 µM [R-32P]-8-azido-ATP, and under these conditions, the same differential labeling phenomenon is observed, although slightly more label is incorporated into P-gp-D555N (Figure 5B).
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ABCB1 p.Asp555Asn 10529234:232:252
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239 As was demonstrated in Figure 5, again, no substantial labeling was observed for P-gp-D555N.
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ABCB1 p.Asp555Asn 10529234:239:86
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251 Crude membrane preparations (50 µg) from HeLa cells infected with vTF7-3 and recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, or P-gp-D555N/ D1200N were photoaffinity-labeled on ice (0 °C) with either (A) 2.5 µM or (B) 77.5 µM [R-32P]-8-azido-ATP.
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ABCB1 p.Asp555Asn 10529234:251:143
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ABCB1 p.Asp555Asn 10529234:251:171
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255 Crude membrane preparations (50-60 µg) from HeLa cells infected with vTF7-3 and recombinant vaccinia viruses expressing wild-type P-gp, P-gp-D555N, P-gp-D1200N, or P-gp-D555N/D1200N were photoaffinity-labeled with either (A) [R-32P]- 8-azido-ATP (2.5 µM) or (B) [125I]IAAP (15 nM) as described in the legend to Figures 4 and 5.
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ABCB1 p.Asp555Asn 10529234:255:146
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ABCB1 p.Asp555Asn 10529234:255:174
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272 Similar to P-gp-D1200N, P-gp-D555N and P-gp-D555N/D1200N were not capable of being vanadate-trapped, confirming their lack of ATPase activity (data not shown).
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ABCB1 p.Asp555Asn 10529234:272:29
status: NEW
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ABCB1 p.Asp555Asn 10529234:272:44
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292 Under the conditions of this assay, P-gp-D555N was labeled poorly, precluding the reliable estimation of the concentration required for 50% inhibition of labeling by ATP for this construct.
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ABCB1 p.Asp555Asn 10529234:292:41
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294 Under the conditions of the assay, the binding capacity of the C-site, as demonstrated by labeling of P-gp-D555N, is limited and could not be reliably quantitated.
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ABCB1 p.Asp555Asn 10529234:294:107
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297 To determine if conformational differences exist between active wild-type Pgp and inactive P-gp-D555N and P-gp-D1200N, the reactivity of these constructs expressed transiently in HeLa cells was assessed by use of the conformation-sensitive monoclonal anti-human P-gp antibody UIC2 (5, 45).
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ABCB1 p.Asp555Asn 10529234:297:96
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300 Furthermore, the UIC2 reactivity of cells expressing either P-gp-D555N and P-gp-D1200N is indistinguishable from each other, in either the presence or absence of cyclosporin A, and is equivalent to that of the cyclosporin A-treated wild-type-expressing cells.
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ABCB1 p.Asp555Asn 10529234:300:65
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301 These UIC2 reactivity data, taken together with the ATPase data that demonstrated complete abrogation of ATPase activity for the D555N and D1200N P-gp proteins (Figure 3) and the ATP labeling data that demonstrated marked differences in the ability of these molecules to bind ATP (Figure 5), strongly suggest that the intrinsic ability of the P-gp molecule to hydrolyze ATP or associate with hydrolysis products accounts for the difference in conformation detected by UIC2.
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ABCB1 p.Asp555Asn 10529234:301:129
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307 The extremely low labeling efficiency of P-gp-D555N precluded analysis of that protein in this assay.
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ABCB1 p.Asp555Asn 10529234:307:46
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315 FIGURE 8: UIC2 reactivity shift induced by incubation with cyclosporin A. vTF7-3-infected-transfected HeLa cells expressing wild-type P-gp, P-gp-D555N, or P-gp-D1200N were subjected to FACS analysis after staining with UIC2 in the presence (thin line) and absence (bold line) of 5 µM cyclosporin A.
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ABCB1 p.Asp555Asn 10529234:315:145
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354 These mutations at positions D555N and D1200N, made separately or in concert, did not affect the ability of the transporter to bind substrate but completely abrogated transport function and both basal and drug-stimulated ATPase activity.
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ABCB1 p.Asp555Asn 10529234:354:29
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365 Whereas P-gp-D1200N, the mutant transporter with a C-terminal amino acid substitution, labels comparably to the wild-type protein, P-gp-D555N, containing a homologous N-terminal mutation, is severely impaired in its ability to be labeled with [R-32P]-8-azido-ATP.
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ABCB1 p.Asp555Asn 10529234:365:136
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366 The inability of P-gp-D555N to be labeled efficiently even though the C-half NBD was intact may be because the mutation severely affected the ability of Mg‚ATP to bind to the N-half NBD, and the protein exists in such a conformation that either the C-half NBD site is obscured from nucleotide or the amino acid to which the azido group is to be cross-linked is improperly positioned.
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ABCB1 p.Asp555Asn 10529234:366:22
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367 This hypothesis that the N-half NBD was impaired for labeling in the D555N protein was supported through analysis of the labeling pattern of the two halves of the molecule.
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ABCB1 p.Asp555Asn 10529234:367:69
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368 After mild trypsinization, it was apparent that the N-half is predominantly labeled in the D1200N and wild-type proteins, whereas little to no labeling of the N-half of the P-gp-D555N was observed.
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ABCB1 p.Asp555Asn 10529234:368:178
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393 This model is also consistent with the data from the nonfunctional Walker B motif mutants, P-gp-D555N and P-gp-D1200N.
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ABCB1 p.Asp555Asn 10529234:393:96
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395 In the case of P-gp-D555N, only the C-half is intact.
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ABCB1 p.Asp555Asn 10529234:395:20
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PMID: 10698967 [PubMed] Lee CG et al: "Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter."
No. Sentence Comment
21 MDR1mt contains the mutant residue Asn in place of Asp at position 555 of the molecule (D555N).
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ABCB1 p.Asp555Asn 10698967:21:35
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ABCB1 p.Asp555Asn 10698967:21:88
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41 Target HeLa cells were coinfected with VCB3 (CD4-expressing vv), VTF7-3 (vv expressing T7 RNA polymerase), and either of the following vv: WR (wild-type control), MDR1, D555N (amino-terminal ATP binding site mutant of MDR1), V185 (substrate mutant of MDR1 that changes substrate specificity of MDR1) (18), or CFTR (19).
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ABCB1 p.Asp555Asn 10698967:41:169
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58 Bicistronic constructs expressing either wild-type (wt) MDR1 or MDR1 mutated (D555N) at the ATP utilization site (mt) to inactivate P-gp pump function and DHFR were stably introduced into 12D7 cells by selection with methotrexate (24, 25).
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ABCB1 p.Asp555Asn 10698967:58:78
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82 Similar results were seen in five independent experiments. Figure 3 shows that fusion was greatly reduced in target cells expressing either wt MDR1, MDR1 with an inactivating amino-terminal ATP-utilization site (D555N) mutation that renders the transporter inactive or MDR1 with a single mutation in the substrate binding site (G185V) (18).
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ABCB1 p.Asp555Asn 10698967:82:214
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96 WR: wild-type control; MDR1: wild-type MDR1; D555N: amino-terminal ATP binding site mutant of MDR1; G185V; substrate mutant of MDR1 that changes the substrate specificity; CFTR: cystic fibrosis transmembrane regulator.
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ABCB1 p.Asp555Asn 10698967:96:45
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152 We wish to thank J. A. Hoxie (University of Pennsylvania) for providing 12G5 CXCR4 antibody; Y. Sugimoto (Japanese Foundation for Cancer Research) for providing biotinylated MRK16-Fab antibody; G. Englund (NIAID) for help with the three-color FACS analyzes; K. Strebel (NIAID) for help with fluorescence microscopy; C. A. Hrycyna (NCI) for providing the D555N cDNA, for further construction of the pHMIDmt, and the generation of recombinant vaccinia viruses; E. A. Berger (NIAID) for providing VCB3, VTF7-3, VCB21, VCB16, and VCB60 vaccinia viruses; M. Welsh (University of Iowa College of Medicine) for the CFTR vaccinia virus; and Y. Raviv and R. Blumenthal (NCI-Frederick) for discussions and data sharing.
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ABCB1 p.Asp555Asn 10698967:152:354
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PMID: 10758006 [PubMed] Julien M et al: "Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity."
No. Sentence Comment
232 Additional photolabeling experiments using the Walker B mutants D555N and D1200N showed that in contrast to results from Urbatsch et al. (15), only the D1200N mutant can be photolabeled under binding conditions with all the label being present at the NBD1 (16).
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ABCB1 p.Asp555Asn 10758006:232:64
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PMID: 10820025 [PubMed] Booth CL et al: "Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein."
No. Sentence Comment
8 Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding.
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ABCB1 p.Asp555Asn 10820025:8:8
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45 pTM1-MDR1 and pTM1-MDR1-D555N were supplied by Dr. Christine Hrycyna (25, 29).
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ABCB1 p.Asp555Asn 10820025:45:24
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62 A plasmid, containing a Table 1: Cloning Summary for Expression Vectors plasmid name cloning vector PCR primers (5' f 3')a restriction enzymesb pET-NBD1MDR1-C431A (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A/D555N (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A (412-574) pET23a A: GTTAAGATGCATATGGGCCTGAACCTGAAGGTGCAGAGT A: NdeI B: ACCTTTGAATTCTCAATCCAGAGCCACCTGAACCACTGC B: EcoRI pET-NBD1MDR1-C431A (358-590) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: ATTGGATCCTCAAGACAAACGATGAGCTATCACAATGGT B: BamHI pET-NBD1MDR1-C431A (358-635) pET23a A: GAGATATACATATGGCAAGAGGAGCA A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (409-635) pET23a A: GAGACATATGATCTTGAAGGGCCTGAACCTG A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (375-707) pET23a A: GAGACATATGATTGACAGCTATTCGAAGAGT A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (392-707) pET23a A: GAGACATATGTTGGAATTCAGAAATGTTCAC A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (348-635) pET23a A: GAGACATATGGCATCTCCAAGCATTGAAGCATTTGCAAAT- GCAAGAGGAGCAGCT A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI a A, forward primer; B, reverse primer.
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ABCB1 p.Asp555Asn 10820025:62:301
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64 sequence encoding MDR1 amino acids 358-707 with both C431A and D555N mutations, was prepared as described above, with the pTM1-MDR1-D555N vector bearing the MDR1 coding sequence with a D555N mutation as the primary template, and was labeled pET-NBD1MDR1-C431A/ D555N (358-707).
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ABCB1 p.Asp555Asn 10820025:64:63
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ABCB1 p.Asp555Asn 10820025:64:132
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ABCB1 p.Asp555Asn 10820025:64:185
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ABCB1 p.Asp555Asn 10820025:64:261
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92 Sample consisted of NBD1 (358-707) or NBD1 D555N (358-707) proteins (~250 µg/mL) in 50 mM potassium phosphate/50 mM NaCl (pH 7.0).
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ABCB1 p.Asp555Asn 10820025:92:43
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129 (A) Expression of NBD1 proteins before (lanes 1, 3, 5, and 7) and after (lanes 2, 4, 6, and 8) IPTG induction. NBD1 proteins were as follows: NBD1 (358-707), lanes 1-2; NBD1 D555N (358-707), lanes 3 and 4; NBD1 (412-574), lanes 5 and 6; and NBD1 (358-590), lanes 7 and 8.
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ABCB1 p.Asp555Asn 10820025:129:174
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130 (B) Purified NBD1 proteins were identified by colloidal blue staining as follows: NBD1 (358-707), lane 1; NBD1 D555N (358-707), lane 2; NBD1 (412-574), lane 3; and NBD1 (358-590), lane 4.
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ABCB1 p.Asp555Asn 10820025:130:111
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133 NBD1 proteins were as follows: (A) NBD1 (358-707), (B) NBD1 (358-590), (C) NBD1 (412-574), and (D) NBD1 D555N (358-707).
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ABCB1 p.Asp555Asn 10820025:133:104
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136 D555N Amino Acid Substitution in the Walker B Consensus Sequence Results in an Alteration in Mg2+ -Enhanced ATP Binding.
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ABCB1 p.Asp555Asn 10820025:136:0
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137 To evaluate the effect of Mg2+ on [R-32 P]-8-azido-ATP labeling, the NBD1 (358-707) protein was mutated at position 555 from aspartate to asparagine in the Walker B consensus motif (36).
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ABCB1 p.Asp555Asn 10820025:137:116
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139 Thus, the NBD1 D555N (358-707) protein serves as a specificity control in the present work.
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ABCB1 p.Asp555Asn 10820025:139:15
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140 Expression and purification of NBD1 D555N (358-707) protein is shown in Figure 1.
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ABCB1 p.Asp555Asn 10820025:140:36
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141 As demonstrated, NBD1 D555N (358-707) protein was localized in the inclusion-body protein and was purified by the methods described previously.
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ABCB1 p.Asp555Asn 10820025:141:22
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142 Unlike the wild-type NBD1 (358-707) protein, [R-32 P]-8-azido-ATP labeling of the mutant NBD1 D555N (358-707) protein was greater in the absence of MgSO4 than in the presence of MgSO4 (Figure 2D).
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ABCB1 p.Asp555Asn 10820025:142:94
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143 In fact, in the presence of MgSO4 the ability of [R-32 P]-8-azido-ATP to label NBD1 D555N (358-707) protein was impaired severely (Figure 2D).
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ABCB1 p.Asp555Asn 10820025:143:84
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144 Incubation of [R-32P]-8-azido-ATP with NBD1 (358-707) protein or NBD1 D555N (358-707) protein in the presence of increasing concentrations of MgSO4 resulted in an increase in labeling with wild-type protein and a decrease in labeling with mutant protein (data not shown).
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ABCB1 p.Asp555Asn 10820025:144:70
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145 The specificity of labeling of NBD1 D555N (358-707) protein was explored by incubating the protein with [R-32 P]-8-azido-ATP in the presence of increasing concentrations of ATP in the absence of MgSO4.
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ABCB1 p.Asp555Asn 10820025:145:36
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148 Analysis of NBD1 (358-707) and NBD1 D555N (358-707) proteins by CD was performed in order to assess the secondary structural components and to evaluate the effect of Mg2+.
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ABCB1 p.Asp555Asn 10820025:148:36
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149 The predicted secondary structural components of these proteins were 25% R-helix, 23% -strand, and 19% -turn; no differences between NBD1 (358-707) and NBD1 D555N (358-707) proteins were demonstrated (data not shown).
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ABCB1 p.Asp555Asn 10820025:149:157
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150 Similarly, the predicted secondary structural elements of NBD1 (358-707) and NBD1 D555N (358-707) proteins in the presence of 2 mM MgSO4 were not different than in the absence of MgSO4 (data not shown).
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ABCB1 p.Asp555Asn 10820025:150:82
status: NEW
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151 A temperature melt was performed to determine the thermal stability of NBD1 (358-707) and NBD1 D555N (358-707) proteins.
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ABCB1 p.Asp555Asn 10820025:151:95
status: NEW
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220 Secondary structural analysis of NBD1 (358-707) and NBD1 D555N (358-707) proteins by circular dichroism showed 25% R-helix, 23% -strand, and 19% -turn with no significant difference in secondary structure between the two proteins.
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ABCB1 p.Asp555Asn 10820025:220:57
status: NEW
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224 More interesting was the lack of secondary structural change of NBD1 (358-707) and NBD1 D555N (358-707) proteins in the presence of Mg2+ .
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ABCB1 p.Asp555Asn 10820025:224:88
status: NEW
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249 The C431A and D555N mutations within the Walker A and Walker B motifs, respectively, are identified by vertical bars.
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ABCB1 p.Asp555Asn 10820025:249:14
status: NEW
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PMID: 11287418 [PubMed] Sauna ZE et al: "Functionally similar vanadate-induced 8-azidoadenosine 5'-[alpha-(32)P]Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis."
No. Sentence Comment
55 Preparation of Crude Membranes from HeLa Cells Expressing Mutant and Wild-type Pgp-A 70-80% confluent monolayer of HeLa cells was infected with vTF7-3 and transfected with pTM1-MDR1 (wild type) or pTM1-MDR1 bearing the homologous mutations at positions 555 (D555N) and 1200 (D1200N) as described previously (9).
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ABCB1 p.Asp555Asn 11287418:55:258
status: NEW
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56 The vaccinia virus expression vectors pTM1-MDR1 (wild type) and pTM1-MDR1(D555N) and pTM1-MDR1(D1200N) were provided by C. A. Hrycyna and M. M. Gottesman, Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda.
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ABCB1 p.Asp555Asn 11287418:56:74
status: NEW
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224 The Pgp mutants D555N and D1200N in Walker B domain of ATP sites do not show Vi-induced trapping of [␣-32 P]8-azido-ADP using either [␣-32 P]8-azido-ADP or [␣-32 P]8-azido-ATP.
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ABCB1 p.Asp555Asn 11287418:224:16
status: NEW
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225 Crude membranes were prepared from HeLa cells transiently expressing the pTM1-MDR1 (designated wild type) and pTM1-MDR1 bearing the homologous mutations in Walker B region of ATP sites at positions 555 and 1200 (designated D555N and D1200N), respectively.
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ABCB1 p.Asp555Asn 11287418:225:223
status: NEW
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230 Lane 1, wild-type Pgp membranes labeled with [␣-32 P]8-azido-ATP; lane 2, D555N membranes labeled with [␣-32 P]8-azido-ATP; lane 3, D1200N membranes labeled with [␣-32 P]8-azido-ATP; lane 4, wild-type membranes labeled with [␣-32 P]8-azido-ADP; lane 5, D555N membranes labeled with [␣-32 P]8-azido-ADP; and lane 6, D1200N membranes labeled with [␣-32 P]8-azido-ADP.
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ABCB1 p.Asp555Asn 11287418:230:81
status: NEW
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ABCB1 p.Asp555Asn 11287418:230:281
status: NEW
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290 The mutations in either the N-terminal ATP site (D555N) or the C-terminal site (D1200N) abolish Vi-induced trapping of [␣-32 P]8-azido-ADP by both the hydrolysis and non-hydrolysis routes (Fig. 6).
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ABCB1 p.Asp555Asn 11287418:290:49
status: NEW
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PMID: 9788623 [PubMed] Russ G et al: "P-glycoprotein plays an insignificant role in the presentation of antigenic peptides to CD8+ T cells."
No. Sentence Comment
67 Further rVVs were used to express P-glycoprotein |MDRI(T7)] and P-glycoprotein mutants (NM- D555N, CM-D1200N, and DM-D555N + D1200N), under the control of the T7 promoter (14).
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ABCB1 p.Asp555Asn 9788623:67:92
status: NEW
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ABCB1 p.Asp555Asn 9788623:67:117
status: NEW
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109 The introduced mutations alter one or both of highly conserved Asp residues located, respectively, within Walker B regions of either NH2-terminal (D555N) or COOH-terminal (D1200N) ATP binding/utilization sites that are believed to be involved in binding to Mg2+.
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ABCB1 p.Asp555Asn 9788623:109:147
status: NEW
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64 Further rVVs were used to express P-glycoprotein |MDRI(T7)] and P-glycoprotein mutants (NM- D555N, CM-D1200N, and DM-D555N + D1200N), under the control of the T7 promoter (14).
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ABCB1 p.Asp555Asn 9788623:64:92
status: NEW
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ABCB1 p.Asp555Asn 9788623:64:117
status: NEW
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105 To determine the requirement for a functional P-glycoprotein, we used rVVs expressing three mutated forms of the protein under the control of the T7 promoter, termed CM, NM, and DM. The introduced mutations alter one or both of highly conserved Asp residues located, respectively, within Walker B regions of either NH2-terminal (D555N) or COOH-terminal (D1200N) ATP binding/utilization sites that are believed to be involved in binding to Mg2+.
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ABCB1 p.Asp555Asn 9788623:105:329
status: NEW
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PMID: 16545467 [PubMed] Shilling RA et al: "New light on multidrug binding by an ATP-binding-cassette transporter."
No. Sentence Comment
58 Although mutation of only one of these residues (L975A, V981A and F983A) has no effect on the phenotype of the protein [20], double mutations either completely inhibit (V981A/F983A and L975A/V981A) or cause 50% inhibition (L975A/F983A) of Table 1.
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ABCB1 p.Asp555Asn 16545467:58:996
status: NEW
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59 Published mutations in human and murine P-glycoprotein that alter drug transport in cells Location of mutation Mutation Refs Mutation Refs Mutation Refs Transmembrane helices H61A and others [14] I214L [60] L868W [59] G64R [15] P223A [65] I936A [21] L65R [15] S224P [60] F938A [21] Q139[H/P/R] [60] I306R [18] S939[A/C/T/Y/W/D/F] [21,22] G141V [17] F335A [16] T941A [21] G185V [61,62] V338A [66] Q942A [21] I186N [61] G338A [67,68] A943G [21] G187V [17] A339P [67,68] Y946A [21] G187E [60] G341A [66] S948A [21] A192T [60] S344[A/T/C/Y] [66] Y949A [21] F200L [60] N350I [19] C952A [21] F204S [60] P709A [65] F953A [21] R206L [60] G830V [17] L975A [20] W208G [60] I837L [23] F978A [16] K209E [60] N839I [23] V981A [20] L210I [60] I862F [19] F983A [20] T211P [60] L865F [19] F978A [16] V213A [60] P866A [65] N988D [59] Intracellular domain T169I [60] K177I [60] G288V [17] R170L [60] E180G [60] A931T [19] L171P [60] G181R [60] F934A [21] T172P [60] G183D [60] G935A [21] S176P [60] D184N [60] NBD D555N [63] K1076M [69] E1197Q [64] D558N [64] D1093N [64] D1203N [64] D592N [64] E1125Q [64] D1237N [64] E604Q [64] S1173A [70] E1249Q [64] Review TRENDS in Pharmacological Sciences Vol.27 No.
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ABCB1 p.Asp555Asn 16545467:59:996
status: NEW
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