ABCG2 p.Asn387Ala
| Predicted by SNAP2: | A: D (71%), C: D (63%), D: D (75%), E: D (80%), F: D (80%), G: D (75%), H: D (75%), I: D (75%), K: D (85%), L: D (80%), M: D (71%), P: D (85%), Q: D (66%), R: D (80%), S: D (66%), T: D (71%), V: D (75%), W: D (91%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Transmembrane helices 1 and 6 of the human breast ... Am J Physiol Cell Physiol. 2010 Nov;299(5):C1100-9. Epub 2010 Aug 25. Ni Z, Bikadi Z, Cai X, Rosenberg MF, Mao Q
Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.
Am J Physiol Cell Physiol. 2010 Nov;299(5):C1100-9. Epub 2010 Aug 25., [PMID:20739628]
Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane alpha-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.
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No. Sentence Comment
12 N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin.
X
ABCG2 p.Asn387Ala 20739628:12:0
status: VERIFIED15 Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity.
X
ABCG2 p.Asn387Ala 20739628:15:62
status: VERIFIED68 The forward primers used for mutagenesis were as follows: N387A (5=- AAG CGT TCA TTC AAA GCC TTG CTG GGT AAT CCC-3=), Q398A (5=-CAG GCC TCT ATA GCT GCG ATC ATT GTC ACA GTC-3=), T402A (5=-GCT CAG ATC ATT GTC GCA GTC GTA CTG GGA CTG-3=), N629A (5=-CTG GGG CTT GTG GAA GGC TCA CGT GGC CTT GGC TTG-3=), T642A (5=-GAT TGT TAT TTT CCT CGC AAT TGC CTA CCT GAA ATT G-3=), and Y645F (5=-TTC CTC ACA ATT GCC TTC CTG AAA TTG TTA TTT C-3=).
X
ABCG2 p.Asn387Ala 20739628:68:58
status: VERIFIED162 The levels of N387A, Q398A, T402A, N629A, T642A, and Y645F, determined by immunoblotting of whole cell lysates using beta-actin as an internal standard, were ϳ4.4-, 4.5-, 3.1-, 0.4-, 1.3-, and 1.5-fold that of wild-type BCRP (Fig. 2, A and B).
X
ABCG2 p.Asn387Ala 20739628:162:14
status: VERIFIED191 N387A and Q398A also showed significantly reduced efflux activities for MX and Hoechst 33342 by 40-80%, but were fully active in transporting BODIPY-prazosin.
X
ABCG2 p.Asn387Ala 20739628:191:0
status: VERIFIED200 Representative areas of HEK-293 cells expressing wild-type BCRP and the mutants N387A, Q398A, T402A, N629A, T642A, and Y645F are shown.
X
ABCG2 p.Asn387Ala 20739628:200:80
status: VERIFIED217 Thus N387A, Q398A, and T402A exhibited a significantly lower resistance to MX than wild-type BCRP, whereas N629A displayed an approximately threefold increase in resistance to MX.
X
ABCG2 p.Asn387Ala 20739628:217:5
status: VERIFIED224 The Km values of N387A, T402A, T642A, and Y645F were comparable to that of wild-type protein; however, the Km values of Q398A and N629A were decreased by ϳ50-60%.
X
ABCG2 p.Asn387Ala 20739628:224:17
status: VERIFIED226 After normalization to the BCRP protein levels, the Vmax values of N387A, Q398A, and T402A were approximately one-half of that of wild-type BCRP, whereas the Vmax value of N629A was increased by ϳ90%.
X
ABCG2 p.Asn387Ala 20739628:226:67
status: VERIFIED228 In contrast, the Vmax/Km values of N387A and T402A were decreased by ϳ50%.
X
ABCG2 p.Asn387Ala 20739628:228:35
status: VERIFIED231 FTC-inhibitable efflux activities of HEK-293 cells stably expressing wild-type and mutant BCRP Mitoxantrone BODIPY-Prazosin Hoechst 33342 ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio ⌬F ⌬F= Ratio Wild-type BCRP 10.6 Ϯ 0.6 10.6 Ϯ 0.6 1.0 12.5 Ϯ 4.3 12.5 Ϯ 4.3 1.0 2150.0 Ϯ 25.5 2150.0 Ϯ 25.5 1.0 N387A 11.3 Ϯ 0.5 2.5 Ϯ 0.1* 0.2 54.5 Ϯ 5.8 12.3 Ϯ 1.3 1.0 5,024.7 Ϯ 570.5 1,131.7 Ϯ 128.5* 0.5 Q398A 28.1 Ϯ 5.2 6.3 Ϯ 1.2* 0.6 69.3 Ϯ 10.6 15.5 Ϯ 2.4 1.2 4,206.7 Ϯ 252.1 941.1 Ϯ 56.4* 0.4 T402A 12.3 Ϯ 2.4 4.0 Ϯ 0.8* 0.4 4.5 Ϯ 1.8 1.5 Ϯ 0.6* 0.1 999.3 Ϯ 352.9 322.4 Ϯ 113.8* 0.1 N629A 19.3 Ϯ 3.7 46.0 Ϯ 8.8* 4.3 12.8 Ϯ 1.5 30.5 Ϯ 3.6* 2.4 2,681.0 Ϯ 370.5 6,383.3 Ϯ 882.1* 3.0 T642A 17.0 Ϯ 0.3 12.9 Ϯ 0.2 1.2 15.4 Ϯ 1.3 11.8 Ϯ 1.0 0.9 1,860.7 Ϯ 462.3 1,420.4 Ϯ 352.9* 0.7 Y645F 16.8 Ϯ 2.3 11.6 Ϯ 1.6 1.1 15.1 Ϯ 4.6 10.4 Ϯ 3.2 0.8 1,358.7 Ϯ 35.5 937.0 Ϯ 24.5* 0.4 Values are means Ϯ SD of 3 independent experiments.
X
ABCG2 p.Asn387Ala 20739628:231:354
status: VERIFIED238 Relative drug resistance of HEK-293 cells expressing wild-type and mutant BCRP MX SN-38 Dox Rho-123 IC50, nM RR (ratio) IC50, nM RR (ratio) IC50, nM RR IC50, nM RR pcDNA control 8.5 Ϯ 0.6 2.3 Ϯ 0.28 125.6 Ϯ 32.0 1,881 Ϯ 168.2 Wild-type BCRP 64.8 Ϯ 3.9 7.6 (1.0) 89.7 Ϯ 5.4 39.0 (1.0) 168.0 Ϯ 24.5 1.3 3,341 Ϯ 267.4 1.8 N387A 54.8 Ϯ 6.3 6.4 (0.2)* 44.8 Ϯ 6.3 19.5 (0.1)* 120.4 Ϯ 31.6 1.0 3,279 Ϯ 436.8 1.7 Q398A 49.9 Ϯ 4.6 5.9 (0.2)* 71.6 Ϯ 4.3 31.1 (0.2)* 173.4 Ϯ 36.5 1.4 2,534 Ϯ 376.5 1.3 T402A 43.3 Ϯ 7.6 5.1 (0.2)* 63.0 Ϯ 5.4 27.4 (0.2)* 129.4 Ϯ 31.4 1.0 2,140 Ϯ 210.7 1.1 N629A 76.5 Ϯ 12.3 9.0 (2.8)* 108.3 Ϯ 12.3 47.1 (2.9)* 164.6 Ϯ 14.8 1.3 3,051 Ϯ 286.5 1.6 T642A 50.0 Ϯ 9.5 5.9 (0.6) 49.9 Ϯ 9.9 21.7 (0.4)* 156.1 Ϯ 20.0 1.2 1,393 Ϯ 221.6 0.7 Y645F 42.8 Ϯ 6.8 5.0 (0.5)* 44.8 Ϯ 6.3 19.5 (0.3)* 132.4 Ϯ 18.4 1.1 1,846 Ϯ 206.2 1.0 The IC50 values shown are means Ϯ SD of 3 independent experiments.
X
ABCG2 p.Asn387Ala 20739628:238:367
status: VERIFIED251 In contrast, N387A and Q398A were associated with a slight decrease in 5D3-phycoerythrin fluorescence.
X
ABCG2 p.Asn387Ala 20739628:251:13
status: VERIFIED254 In particular, significant differences in 5D3 binding were observed between wild-type BCRP and the mutants N387A, Q398A, T402A, and N629A at certain prazosin concentrations (Fig. 5).
X
ABCG2 p.Asn387Ala 20739628:254:107
status: VERIFIED274 Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP Wild-type BCRP N387A Q398A T402A N629A T642A Y645F Vmax, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 16.9 Ϯ 1.5 14.9 Ϯ 0.93 17.4 Ϯ 1.7 11.5 Ϯ 1.1 17.9 Ϯ 1.9 14.5 Ϯ 1.9 Vmax normalized to BCRP protein level, nmol Pi ·min-1 ·mg protein-1 14.5 Ϯ 1.9 7.0 Ϯ 0.63 6.5 Ϯ 0.41 8.3 Ϯ 0.81 28.0 Ϯ 2.7 16.3 Ϯ 1.7 12.0 Ϯ 1.6 Km for ATP, mM 0.89 Ϯ 0.40 0.90 Ϯ 0.27 0.48 Ϯ 0.12 1.1 Ϯ 0.35 0.40 Ϯ 0.15 0.93 Ϯ 0.34 0.89 Ϯ 0.43 Vmax/Km, nmol Pi ·min-1 ·mg protein-1 ·mM-1 16.3 7.8 13.5 7.5 70.0 17.5 13.5 Values are means Ϯ SD of 3 independent determinations.
X
ABCG2 p.Asn387Ala 20739628:274:81
status: VERIFIED282 Significant differences (P Ͻ 0.05) in 5D3 binding analyzed by the Student`s t-test were observed between wild-type BCRP and the mutant N387A, Q398A, T402A, or N629A at certain prazosin concentrations and are marked (*) on the right of respective data points.
X
ABCG2 p.Asn387Ala 20739628:282:141
status: VERIFIED318 C1107POLAR Replacement of Asn387 and Gln398 with Ala significantly but selectively impaired the efflux of MX and Hoechst 33342, but not of BODIPY-prazosin, and showed lower resistance to MX and SN-38 (Tables 1 and 2), suggesting that mutations of the two polar residues influenced substrate specificity.
X
ABCG2 p.Asn387Ala 20739628:318:27
status: VERIFIEDX
ABCG2 p.Asn387Ala 20739628:318:97
status: NEW323 However, we showed that mutation of Asn387 with Ala did not impair BCRP expression, folding, or trafficking to the plasma membrane to any significant extent.
X
ABCG2 p.Asn387Ala 20739628:323:36
status: VERIFIED342 N387A, Q398A, and T402A also showed changes in Km and/or Vmax for ATP hydrolysis (Table 3).
X
ABCG2 p.Asn387Ala 20739628:342:0
status: VERIFIED350 Prazosin differentially increased 5D3 binding to wild-type BCRP, N629A, T642A, and Y645F, whereas 5D3 binding to N387A and Q398A was slightly decreased (Fig. 5).
X
ABCG2 p.Asn387Ala 20739628:350:113
status: VERIFIED