ABCG2 p.Lys473Ala
| Predicted by SNAP2: | A: D (75%), C: D (71%), D: D (85%), E: D (85%), F: D (85%), G: D (75%), H: D (71%), I: D (80%), L: D (75%), M: D (66%), N: D (66%), P: D (91%), Q: D (66%), R: D (66%), S: D (71%), T: D (71%), V: D (80%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: 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] Role of basic residues within or near the predicte... J Pharmacol Exp Ther. 2010 Jun;333(3):670-81. Epub 2010 Mar 4. Cai X, Bikadi Z, Ni Z, Lee EW, Wang H, Rosenberg MF, Mao Q
Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.
J Pharmacol Exp Ther. 2010 Jun;333(3):670-81. Epub 2010 Mar 4., [PMID:20203106]
Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.
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No. Sentence Comment
2 Lys452 , Lys453 , His457 , Arg465 , and Lys473 were replaced with Ala or Asp.
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ABCG2 p.Lys473Ala 20203106:2:40
status: VERIFIED7 The transport activities and drug-resistance profiles of K473A were not changed.
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ABCG2 p.Lys473Ala 20203106:7:57
status: VERIFIED76 The polymerase chain reaction-based mutagenesis was performed according to the manufacturer`s instructions with the following forward primers: K452A (5Ј-gaa ctc ttt gtg gta gag GCg aag ctc ttc ata cat gaa-3Ј), K453D (5Ј-ctc ttt gtg gta gag aag GaC ctc ttc ata cat gaa tac-3Ј), H457A (5Ј-gag aag aag ctc ttc ata GCt gaa tac atc agc gga tac-3Ј), R465A (5Ј-tac atc agc gga tac tac GCa gtg tca tct tat ttc ctt-3Ј), and K473A (5Ј-tca tct tat ttc ctt gga GCa ctg tta tct gat tta tta-3Ј).
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ABCG2 p.Lys473Ala 20203106:76:463
status: VERIFIED177 To evaluate the role of these basic residues in BCRP activity, we generated mutants in which Lys452 , His457 , Arg465 , and Lys473 were replaced with Ala.
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ABCG2 p.Lys473Ala 20203106:177:124
status: VERIFIED183 The expression levels of the mutants K452A, K453D, H457A, R465A, and K473A, determined by immunoblotting of whole-cell lysates using beta-actin as an internal standard, were approximately 0.74-, 2.56-, 0.24-, 3.87-, and 1.56-fold that of wild-type BCRP (Fig. 2, A and B).
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ABCG2 p.Lys473Ala 20203106:183:69
status: VERIFIED191 A, a representative immunoblot of whole-cell lysates for wild-type BCRP and the mutants K452A, K453D, H457A, R465A, and K473A.
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ABCG2 p.Lys473Ala 20203106:191:120
status: VERIFIED208 Efflux activities of K473A were not significantly changed.
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ABCG2 p.Lys473Ala 20203106:208:21
status: VERIFIED220 After normalization to the BCRP levels, the IC50 values of cells expressing K452A, K453D, H457A, and R465A for MX and SN-38 were significantly different from those of cells expressing wild-type BCRP, whereas the IC50 values of cells expressing K473A and wild-type protein were comparable.
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ABCG2 p.Lys473Ala 20203106:220:244
status: VERIFIED232 The Km values of K453D, R465A, and K473A were comparable with that of wild-type protein; however, the Km values of K452A and H457A were decreased by approximately 50 and 70%, respectively, suggesting that these two mutations, particularly the one at position 457, increased the binding affinity of ATP to BCRP.
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ABCG2 p.Lys473Ala 20203106:232:35
status: VERIFIED235 As a result, the Vmax/Km values of K452A and H457A were increased approximately 210 µm 10 µm 10 µm Wild-type BCRP K452A K453D 10 µm 10 µm10 µm 10 µm H457A R465A K473A Fig. 3.
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ABCG2 p.Lys473Ala 20203106:235:196
status: VERIFIED241 Selected areas of HEK cells expressing wild-type BCRP and the mutants K452A, K453D, H457A, R465A, and K473A are shown.
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ABCG2 p.Lys473Ala 20203106:241:102
status: VERIFIED245 The Vmax/Km value of K473A was comparable with that of wild-type protein.
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ABCG2 p.Lys473Ala 20203106:245:21
status: VERIFIED263 Mitoxantrone BODIPY-Prazosin Hoechst33342 ⌬F ⌬FЈ Ratio ⌬F ⌬FЈ Ratio ⌬F ⌬FЈ Ratio pcDNA vector 0 0 0 0 0 0 Wild-type BCRP 11.1 Ϯ 1.5 11.1 Ϯ 1.5 1.0 49.9 Ϯ 13.1 49.9 Ϯ 13.1 1.0 976.5 Ϯ 115.5 976.5 Ϯ 115.5 1.0 K452A 22.0 Ϯ 5.9 29.7 Ϯ 7.9* 2.7 76.6 Ϯ 22.5 103.5 Ϯ 30.4* 2.1 1138.3 Ϯ 134.7 1538.3 Ϯ 182.1* 1.6 K453D 19.1 Ϯ 5.9 7.5 Ϯ 3.3* 0.7 57.7 Ϯ 15.6 22.6 Ϯ 6.1* 0.4 1116.9 Ϯ 132.2 436.3 Ϯ 51.6* 0.4 H457A 4.5 Ϯ 2.1 18.7 Ϯ 8.7* 1.7 40.1 Ϯ 9.7 167.0 Ϯ 40.2* 3.3 1259.5 Ϯ 343.2 5247.9 Ϯ 1429.9* 5.4 R465A 26.1 Ϯ 3.0 6.8 Ϯ 0.8* 0.6 96.5 Ϯ 16.0 24.9 Ϯ 4.1* 0.5 2217.8 Ϯ 255.2 573.1 Ϯ 65.9* 0.6 K473A 22.2 Ϯ 5.0 14.3 Ϯ 3.2 1.3 83.2 Ϯ 17.6 53.3 Ϯ 11.3 1.1 1411.5 Ϯ 166.8 887.7 Ϯ 104.9 0.9 no effect on phycoerythrin fluorescence.
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ABCG2 p.Lys473Ala 20203106:263:828
status: VERIFIED266 In contrast, prazosin only slightly decreased, rather than increased, the binding of 5D3 to K473A at low concentrations.
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ABCG2 p.Lys473Ala 20203106:266:92
status: VERIFIED268 Limited Trypsin Digestion of Wild-Type BCRP and the Mutants H457A and K473A.
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ABCG2 p.Lys473Ala 20203106:268:70
status: VERIFIED270 Therefore, to provide additional evidence of conformational changes in the BCRP mutants, we performed limited trypsin digestion of plasma membrane preparations of wild-type BCRP and two representative mutants H457A and K473A that showed significant changes in the pattern of 5D3 binding (Fig. 6).
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ABCG2 p.Lys473Ala 20203106:270:219
status: VERIFIED273 In contrast, significant trypsin cleavage of both H457A and K473A began to occur at a trypsin/protein ratio of 1:100 (Fig. 7, B and C).
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ABCG2 p.Lys473Ala 20203106:273:60
status: VERIFIED289 Vanadate-sensitive ATPase activities of wild-type and mutant BCRP were measured with plasma membrane preparations over an ATP concentration range of 0 to 5 mM as described. Shown are means Ϯ S.D. of three independent experiments for wild-type BCRP (f), K452A (), K453D (F), H457A (‚), R465A (ࡗ), and K473A (छ).
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ABCG2 p.Lys473Ala 20203106:289:325
status: VERIFIED297 MX SN-38 Dox Rho123 IC50 Relative Resistance (Ratio) IC50 Relative Resistance (Ratio) IC50 Relative Resistance IC50 Relative Resistance nM nM nM M pcDNA vector 24.0 Ϯ 3.5 2.4 Ϯ 0.3 24.0 Ϯ 8.7 7.26 Ϯ 1.15 Wild-type BCRP 145.1 Ϯ 52.8 6.0 (1.0) 125.3 Ϯ 6.1 52.2 (1.0) 31.5 Ϯ 12.6 1.3 10.97 Ϯ 1.84 1.5 K452A 354.8 Ϯ 68.6 14.8 (3.3)* 103.0 Ϯ 12.5 42.9 (1.1)* 24.2 Ϯ 4.4 1.0 9.27 Ϯ 1.75 1.3 K453D 244.0 Ϯ 99.9 10.2 (0.7)* 136.2 Ϯ 9.9 56.8 (0.4)* 34.0 Ϯ 6.7 1.4 8.22 Ϯ 0.97 1.1 H457A 71.9 Ϯ 12.8 3.0 (2.1)* 90.6 Ϯ 4.6 37.8 (3.0)* 21.8 Ϯ 16.6 0.9 7.61 Ϯ 1.29 1.0 R465A 169.1 Ϯ 49.0 7.0 (0.3)* 224.2 Ϯ 39.7 93.4 (0.5)* 37.9 Ϯ 17.5 1.6 14.65 Ϯ 1.26 2.0 K473A 243.1 Ϯ 114.0 10.1 (1.1) 188.0 Ϯ 19.2 78.3 (0.9) 36.0 Ϯ 10.1 1.5 15.11 Ϯ 1.43 2.1 not Lys452 , Lys453 , and Lys473 , seem to directly participate in the binding of all four substrates.
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ABCG2 p.Lys473Ala 20203106:297:795
status: VERIFIED317 Wild-type BCRP K452A K453D H457A R465A K473A Vmax (nmol Pi/min/mg protein) 18.4 Ϯ 1.8 16.4 Ϯ 1.9 15.1 Ϯ 1.4 3.94 Ϯ 0.07 15.8 Ϯ 2.6 17.1 Ϯ 1.3 Vmax normalized to BCRP level (nmol Pi/min/mg protein) 18.4 Ϯ 1.8 18.4 Ϯ 2.1 7.4 Ϯ 0.7 19.6 Ϯ 0.3 6.7 Ϯ 1.1 17.6 Ϯ 1.3 Km for ATP (mM) 0.69 Ϯ 0.21 0.32 Ϯ 0.15 0.85 Ϯ 0.12 0.17 Ϯ 0.07 0.46 Ϯ 0.11 0.52 Ϯ 0.13 Vmax/Km (nmol Pi/min/mg protein/mM) 26.7 57.5 8.7 115.3 14.6 33.8 0 25 50 75 100 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 Prazosin (µM) ∆F/F0 Fig. 6.
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ABCG2 p.Lys473Ala 20203106:317:39
status: VERIFIED319 The concentration-dependent effects of prazosin on the binding of 5D3 to wild-type and mutant BCRP over a concentration range of 0 to 100 M were determined by using flow cytometry as described. Shown are means Ϯ S.D. of three independent experiments for the pcDNA control (f), wild-type BCRP (Œ), K452A (छ), K453D (ࡗ), H457A (F), R465A (Ⅺ), and K473A (‚).
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ABCG2 p.Lys473Ala 20203106:319:384
status: VERIFIED320 1 2 3 4 5 6 7 8 9 Wild-type BCRP 72 43 1 2 3 4 5 6 7 8 9 kDa 34 BCRP fraction: 1.0 1.0 0.9 0.9 0.9 0.7 0.6 0.09 0 72 1 2 3 4 5 6 7 8 9 H457A kDa 43 34 1 2 3 4 5 6 7 8 9 BCRP fraction: 1.0 1.4 1.3 1.3 1.5 1.4 0.5 0 0 K473A kDa 72 43 kDa 34 BCRP fraction: 1.0 1.2 1.0 0.9 0.9 0.9 0.8 0.05 0 Fig. 7.
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ABCG2 p.Lys473Ala 20203106:320:216
status: VERIFIED321 Trypsin digestion of wild-type BCRP, H457A, and K473A.
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ABCG2 p.Lys473Ala 20203106:321:48
status: VERIFIED322 Plasma membrane preparations expressing wild-type BCRP, H457A, or K473A (2 g of protein each lane) were subjected to limited trypsin digestion and immunoblotting as described under Materials and Methods.
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ABCG2 p.Lys473Ala 20203106:322:66
status: VERIFIED331 Conversion of Lys473 to Ala had no significant effect on BCRP activity.
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ABCG2 p.Lys473Ala 20203106:331:14
status: VERIFIED375 Prazosin differentially increased 5D3 binding to wild-type BCRP, K452A, K453D, H457A, and R465A, but had little effect on 5D3 binding to K473A (Fig. 6).
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ABCG2 p.Lys473Ala 20203106:375:137
status: VERIFIED379 K473A appears to be the case that revealed a completely different pattern of 5D3 binding (Fig. 6) and increased resistance to trypsin digestion (Fig. 7) compared with wild-type protein, but showed no activity changes.
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ABCG2 p.Lys473Ala 20203106:379:0
status: VERIFIED[hide] Structure and function of the human breast cancer ... Curr Drug Metab. 2010 Sep;11(7):603-17. Ni Z, Bikadi Z, Rosenberg MF, Mao Q
Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).
Curr Drug Metab. 2010 Sep;11(7):603-17., [PMID:20812902]
Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.
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No. Sentence Comment
314 Such mutants include K473A and H630X, suggesting that these residues are likely not critical for expression and function of BCRP.
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ABCG2 p.Lys473Ala 20812902:314:21
status: VERIFIED[hide] Identification of residues in ABCG2 affecting prot... Biosci Rep. 2015 Jul 17;35(4). pii: e00241. doi: 10.1042/BSR20150150. Haider AJ, Cox MH, Jones N, Goode AJ, Bridge KS, Wong K, Briggs D, Kerr ID
Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.
Biosci Rep. 2015 Jul 17;35(4). pii: e00241. doi: 10.1042/BSR20150150., [PMID:26294421]
Abstract [show]
ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). 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.
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No. Sentence Comment
109 We made individual substitutions of each amino acid to alanine: M131A, S195A, K453A, K473A, P485A, M549A, W564A and I573A.
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ABCG2 p.Lys473Ala 26294421:109:85
status: NEW153 Firstly, there was a group of five mutants which showed FTC-inhibited MX export comparable to WT protein (e.g. M131A, S195A, K453A, K473A, W564A; M131A data shown in Figure 3C).
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ABCG2 p.Lys473Ala 26294421:153:132
status: NEW