ABCMdb

ABCG2 p.Thr402Ala

Predicted by SNAP2:	A: D (53%), C: D (71%), D: D (53%), E: D (66%), F: D (63%), G: D (63%), H: D (53%), I: D (59%), K: D (75%), L: D (71%), M: N (53%), N: N (66%), P: D (59%), Q: D (63%), R: D (75%), S: N (66%), V: D (59%), W: D (85%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: N, P: D, Q: D, R: D, S: N, V: N, W: D, Y: D,

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Publications
[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
9 We substituted Asn387 , Gln398 , Asn629 , and Thr642 with Ala, Thr402 with Ala and Arg, and Tyr645 with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Login to comment
11 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. Login to comment
15 Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Login to comment
68 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=). Login to comment
82 Generation of Flp-In-293 cells stably expressing wild-type BCRP and the mutants T402A and T402R. Login to comment
85 The resulting pcDNA5/FRT/BCRP plasmid containing full-length human BCRP cDNA was used as a template to generate T402A and T402R, as described above. Login to comment
87 The forward primer used to generate T402A was the same as described. Login to comment
88 One microgram of the pcDNA5/ FRT plasmid containing wild-type BCRP, T402A, or T402R cDNA or the empty vector were used to transfect Flp-In-293 cells, in combination with 2 ␮g of pOG44 using the FuGENE HD transfection reagent, according to the manufacturer`s instruction. Login to comment
90 The cells expressing wild-type BCRP, T402A, or T402R were then maintained in complete MEM medium containing 10% FBS, L-glutamine, and 125 ␮g/ml of hygromycin for subsequent experiments. Login to comment
162 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). Login to comment
188 However, HEK-293 cells expressing wild-type and mutant BCRP showed substantial efflux activities for all of the three substrates tested, with the exception of T402A for BODIPY-prazosin, suggesting that mutation of Thr402 drastically impaired the ability of BCRP to transport BODIPY-prazosin. Login to comment
190 However, T402A showed significantly decreased efflux activities for all the three substrates by 60-90%, with particularly lower efflux activities for BODIPY-prazosin and Hoechst 3342. Login to comment
200 Representative areas of HEK-293 cells expressing wild-type BCRP and the mutants N387A, Q398A, T402A, N629A, T642A, and Y645F are shown. Login to comment
217 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. Login to comment
224 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%. Login to comment
226 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%. Login to comment
228 In contrast, the Vmax/Km values of N387A and T402A were decreased by ϳ50%. Login to comment
231 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. Login to comment
238 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. Login to comment
252 The pattern of 5D3 binding to T402A fell between the above two groups. Login to comment
254 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). Login to comment
255 Efflux activities of the mutants T402A and T402R expressed in Flp-In-293 cells. Login to comment
256 In the experiments described above, the protein expression level of T402A in HEK-293 cells was approximately three times greater than that of wild-type BCRP (Fig. 2). Login to comment
257 To investigate whether the decreased efflux activity of T402A was caused by altered pattern of oligomerization resulting from the higher level of protein expression that could subsequently affect BCRP activity, we generated Flp-In-293 cells stably expressing wild-type BCRP, T402A, or T402R. Login to comment
261 Indeed, as shown in Fig. 6A, wild-type BCRP, T402A, and T402R were expressed at comparable protein levels. Login to comment
262 Immunofluorescent confocal microscopy analysis indicated that T402A and T402R were predominantly localized on the plasma membrane of Flp-In-293 cells as was wild-type BCRP (Fig. 6B). Login to comment
263 Consistent with the efflux data of T402A expressed in regular HEK-293 cells, the mutant expressed in Flp-In-293 cells also showed significantly decreased efflux activities for MX, BODIPY-prazosin, and Hoechst 33342 (Fig. 6C). Login to comment
265 Interestingly, efflux activities of T402R were further decreased compared with T402A, suggesting that Arg substitution of Thr402 caused a greater effect on BCRP function than Ala substitution, possibly due to more drastic changes in both size and polarity at position 402. Login to comment
274 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. Login to comment
282 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. Login to comment
290 We verified that T402A and T402R, which were expressed in Flp-In-293 cells at comparable protein levels to wild-type BCRP, also exhibited significantly decreased efflux activities (Fig. 6). Login to comment
300 Expression and efflux activities of T402A and T402R in Flp-In-293 cells. Login to comment
301 A: representative immunoblot of whole cell lysates for wild-type BCRP and its mutants T402A and T402R expressed in Flp-In-293 cells. Login to comment
303 B: confocal microscopy analysis of Flp-In-293 cells expressing wild-type BCRP and its mutants T402A and T402R. Login to comment
305 C: fumitremorgin C (FTC)-inhibitable efflux activities of Flp-In-293 cells expressing wild-type BCRP and its mutants T402A and T402R for mitoxantrone, BODIPY-prazosin, and Hoechst 33342. Login to comment
342 N387A, Q398A, and T402A also showed changes in Km and/or Vmax for ATP hydrolysis (Table 3). Login to comment
351 Prazosin only moderately increased 5D3 binding to T402A (Fig. 5). Login to comment