ABCG2 p.Pro485Ala
[switch to full view]Comments [show]
None has been submitted yet.
PMID: 21854076
[PubMed]
Ni Z et al: "Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity."
No.
Sentence
Comment
4
While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin.
X
ABCG2 p.Pro485Ala 21854076:4:128
status: NEW7 ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP.
X
ABCG2 p.Pro485Ala 21854076:7:60
status: NEW9 Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner.
X
ABCG2 p.Pro485Ala 21854076:9:117
status: NEW46 Full-length wild-type human BCRP cDNA in pcDNA3.1 was subcloned into pcDNA5/ FRT.15 The pcDNA5/FRT plasmid containing full-length human BCRP cDNA was used as a template to generate all the BCRP mutants using PCR mutagenesis as previously described.15 The following forward primers were used for mutagenesis: P392A (5'-AAC TTG CTG GGT AAT GCC CAG GCC TCT ATA GCT C-3'), P480A (5'-CTG TTA TCT GAT TTA TTA GCC ATG AGG ATG TTA CCA AG-3'), P485A (5'-CCC ATG AGG ATG TTA GCA AGT ATT ATA TTT ACC TGT ATA G-3'), P501A (5'-CAT GTT AGG ATT GAA GGC AAA GGC AGA TGC CTT C-3'), P574A (5'-CAG TAC TTC AGC ATT GCA CGA TAT GGA TTT ACG-3'), and P623A (5'-GGC ATC GAT CTC TCA GCC TGG GGC TTG TGG AAG AAT-3').
X
ABCG2 p.Pro485Ala 21854076:46:435
status: NEW79 (D) Protein expression levels of P392A and P485A relative to that of wild-type BCRP (100%) in the plasma membrane samples.
X
ABCG2 p.Pro485Ala 21854076:79:43
status: NEW104 The estimated relative cell surface expression levels of wild-type and mutant BCRP based on the differences in median fluorescence between 5D3 and IgG2b control incubations were 100 ± 22, 108 ± 4, 89 ± 1, 139 ± 32, 75 ± 16, 76 ± 9, and 114 ± 0.7% for three independent determinations for wild-type BCRP, P392A, P480A, P485A, P501A, P574A, and P623A, respectively.
X
ABCG2 p.Pro485Ala 21854076:104:353
status: NEW106 The 5D3 fluorescence associated with P485A was increased by 39% compared to that of wild-type BCRP, but the difference was not statistically significant (p > 0.05 by a Figure 3.
X
ABCG2 p.Pro485Ala 21854076:106:37
status: NEW129 The pcDNA5 vector control, wild-type BCRP, P392A, P480A, P485A, P501A, P574A, and P623A are indicated by P, WT, 392, 480, 485, 501, 574, and 623, respectively.
X
ABCG2 p.Pro485Ala 21854076:129:57
status: NEW132 Drug Resistance Profile of Wild-Type and Mutant BCRPa MX SN-38 Dox Rho123 IC50 (nM) RR IC50 (nM) RR IC50 (nM) RR IC50 (nM) RR pcDNA5/FRT 5.6 ± 0.8 - 1.7 ± 0.1 - 33.1 ± 2.2 - 4218 ± 400.0 - wild-type BCRP 51.5 ± 8.5 9.2 30.3 ± 2.0 17.8 40.9 ± 6.7 1.2 4446 ± 261.5 1.1 P392A 24.6 ± 3.8 4.4* 13.1 ± 1.0 7.7* 34.8 ± 1.7 1.1 5326 ± 414.1 1.3 P480A 69.4 ± 10.5 12.4 37.8 ± 1.5 22.2 29.0 ± 4.0 0.9 5107 ± 272.7 1.2 P485A 38.5 ± 7.0 6.9 15.9 ± 2.6 9.4* 41.8 ± 5.3 1.3 3237 ± 199.5 0.8 P501A 40.9 ± 14.6 7.3 27.5 ± 2.4 16.2 23.2 ± 3.8 0.7 4480 ± 816.4 1.1 P574A 49.6 ± 12.5 8.9 32.5 ± 2.6 19.1 29.6 ± 3.5 0.9 5320 ± 419.8 1.3 P623A 68.4 ± 14.4 12.2 36.8 ± 2.8 21.6 37.0 ± 8.8 1.1 3302 ± 388.1 0.8 a The relative resistance (RR) values represent the relative levels of resistance of the mutants compared to that of wild-type BCRP and were calculated by dividing the IC50 values of wild-type and mutant BCRP by the IC50 values of the vector control.
X
ABCG2 p.Pro485Ala 21854076:132:488
status: NEW137 On the other hand, both P392A and P485A showed significantly decreased resistance to SN-38 compared to that of the wild type, thus providing additional evidence that alanine substitution of Pro485 changed the substrate specificity of BCRP.
X
ABCG2 p.Pro485Ala 21854076:137:34
status: NEWX
ABCG2 p.Pro485Ala 21854076:137:166
status: NEW142 To examine if the changes in efflux activity of mutant BCRP are caused by alterations in its ability to hydrolyze ATP, we measured vanadate-sensitive ATPase activities of wild-type BCRP, P392A, and P485A in plasma membrane preparations.
X
ABCG2 p.Pro485Ala 21854076:142:198
status: NEW143 The protein expression levels of wild-type BCRP, P392A, and P485A in plasma membranes were determined by immunoblotting using mAb BXP-21 and found to be comparable after normalization to the internal plasma membrane control Na+ / K+ -ATPase (Figure 2C,D).
X
ABCG2 p.Pro485Ala 21854076:143:60
status: NEW146 P485A exhibited a modest decrease in ATPase activity compared to that of wild-type BCRP; however, the differences were not statistically significant (Figure 5).
X
ABCG2 p.Pro485Ala 21854076:146:0
status: NEW150 We have previously shown that prazosin binding-induced conformational changes as monitored by measuring the level of binding of 5D3 to BCRP could be affected by single mutations, thereby leading to altered transport activity.15 To investigate whether the mutations of Pro392 and Pro485 cause conformational changes in BCRP that affect transport activity, we measured the level of binding of 5D3 to wild-type BCRP, P392A, or P485A in the presence and absence of varying concentrations of the substrate prazosin (0-40 μM) or MX (0-80 μM).
X
ABCG2 p.Pro485Ala 21854076:150:424
status: NEW153 In the presence of prazosin, wild-type BCRP was associated with a concentration-dependent increase in 5D3-phycoerythrin fluorescence by up to 70%; however, there was an only slight increase in 5D3-phycoerythrin fluorescence for P392A and a slight decrease in 5D3-phycoerythrin fluorescence for P485A (Figure 6A).
X
ABCG2 p.Pro485Ala 21854076:153:294
status: NEW155 The patterns of 5D3-phycoerythrin fluorescence with MX for wild-type BCRP, P392A, and P485A were similar, but different from those with prazosin (Figure 6B).
X
ABCG2 p.Pro485Ala 21854076:155:86
status: NEW162 Concentration-dependent effects of prazosin and MX on the binding of 5D3 to wild-type BCRP, P392A, and P485A.
X
ABCG2 p.Pro485Ala 21854076:162:103
status: NEW164 Shown are means ± SD of three experiments. Asterisks indicate statistically significant differences between wild-type BCRP and P392A or P485A (p < 0.05 by a Student`s t test) at various prazosin concentrations.
X
ABCG2 p.Pro485Ala 21854076:164:141
status: NEW180 We found that alanine substitution of Pro485 had a profound impact on the substrate selectivity of BCRP.
X
ABCG2 p.Pro485Ala 21854076:180:14
status: NEW181 P485A exhibited a significant decrease in the efflux activity of BODIPY-prazosin, but not of MX and Hoechst 33342, and conferred lower resistance to SN-38, but not to MX (Figure 4 and Table 1).
X
ABCG2 p.Pro485Ala 21854076:181:0
status: NEW184 Such changes in transport activity of P392A and P485A do not seem to be related to alterations in Figure 7.
X
ABCG2 p.Pro485Ala 21854076:184:48
status: NEW205 Alanine substitution of Pro485 would eliminate the helical distortion of TM3 and the flexibility and influence the helical packing with neighboring TM helices, thereby affecting transport activity for some substrates.
X
ABCG2 p.Pro485Ala 21854076:205:0
status: NEW206 The results of this study suggest that alanine substitution of Pro485 does not affect binding or transport for MX and Hoechst 33342 but weakens binding and transport for prazosin and SN-38.
X
ABCG2 p.Pro485Ala 21854076:206:39
status: NEW225 Second, the responses of wild-type BCRP, P392A, and P485A to 5D3 binding in the presence of prazosin were significantly different from each other (Figure 6A).
X
ABCG2 p.Pro485Ala 21854076:225:52
status: NEW226 However, there was no difference between wild-type BCRP, P392A, and P485A in response to 5D3 binding in the presence of MX (Figure 6B).
X
ABCG2 p.Pro485Ala 21854076:226:68
status: NEW
PMID: 25236865
[PubMed]
Mao Q et al: "Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update."
No.
Sentence
Comment
190
We found that Ala substitution of Pro485 in TM3 significantly reduced efflux of BODIPY-prazosin by 70%, but had no effect on efflux of mitoxantrone and Hoechst 33342 (94).
X
ABCG2 p.Pro485Ala 25236865:190:14
status: NEW
PMID: 26294421
[PubMed]
Haider AJ et al: "Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences."
No.
Sentence
Comment
8
Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2.
X
ABCG2 p.Pro485Ala 26294421:8:21
status: NEW109 We made individual substitutions of each amino acid to alanine: M131A, S195A, K453A, K473A, P485A, M549A, W564A and I573A.
X
ABCG2 p.Pro485Ala 26294421:109:92
status: NEW154 Secondly, there were a pair of mutant isoforms which showed little or no FTC-mediated inhibition of MX export (i.e. having high accumulation of MX even in the absence of FTC) but normal ABCG2 surface protein expression (P485A and M549A; P485A data shown in Figure 5D).
X
ABCG2 p.Pro485Ala 26294421:154:220
status: NEWX
ABCG2 p.Pro485Ala 26294421:154:237
status: NEW156 To investigate further the two TMD mutations with impaired drug transport (M549A and P485A), we generated stable expressing cell lines expressing sfGFP-tagged isoforms.
X
ABCG2 p.Pro485Ala 26294421:156:85
status: NEW165 Data are representative of at least four independent transfections and reflect negative control data (empty vector, A), positive control data (WT ABCG2 expression, B), a mutant with normal MX transport function (M131A, C) and a mutant with abrogated function but normal surface expression (P485A, D).
X
ABCG2 p.Pro485Ala 26294421:165:290
status: NEW171 sfGFP-P485A retained limited MX transport function with a 1.6-fold increase in accumulation in the presence of Ko143.
X
ABCG2 p.Pro485Ala 26294421:171:6
status: NEW174 In contrast, both sfGFP-ABCG2-WT and sfGFP-P485A were able to export PhA in a Ko143-inhibitable manner, with indistinguishable levels of Ko143 inhibition (Figure 6B), indicating positional specific effects on drug binding and transport.
X
ABCG2 p.Pro485Ala 26294421:174:43
status: NEW179 Of the eight residues we mutated, two resulted in altered drug export function (P485A and M549A) and a further residue (I573A) perturbs the trafficking and maturation of ABCG2 glycosylation.
X
ABCG2 p.Pro485Ala 26294421:179:80
status: NEW181 Intriguingly, two of the residues we have analysed (I573 and P485A) are coupled to a residue Ser195 , which can be mutated to alanine without any apparent affect.
X
ABCG2 p.Pro485Ala 26294421:181:61
status: NEW193 Two of the remaining residues we mutated (M549A and P485A) affected drug export and provide some further evidence for a role for TM3 in forming part of a drug-binding site on ABCG2.
X
ABCG2 p.Pro485Ala 26294421:193:52
status: NEW200 A previous study [42] also showed P485A mutation resulted in altered substrate specificity (although the majority of MX transport was retained in that study [42]).
X
ABCG2 p.Pro485Ala 26294421:200:34
status: NEW206 9 to determine whether the lack of function of the P485A and M549A isoforms is due to impaired substrate binding (i.e. a direct effect on the drug-binding site) and/or impaired TMD-NBD communication.
X
ABCG2 p.Pro485Ala 26294421:206:53
status: NEW