ABCMdb

ABCG2 p.Arg465Ala

Predicted by SNAP2:	A: D (80%), C: D (75%), D: D (95%), E: D (91%), F: D (91%), G: D (75%), H: D (66%), I: D (91%), K: D (80%), L: D (91%), M: D (85%), N: D (85%), P: D (95%), Q: D (85%), S: D (75%), T: D (80%), V: D (85%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: 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
3 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. Login to comment
5 After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Login to comment
6 Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. Login to comment
8 These mutations also differentially affected BCRP ATPase activities with a 2to 4-fold increase in Vmax/Km for K452A and H457A and a 40 to 70% decrease for K453D and R465A. Login to comment
76 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). Login to comment
183 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). Login to comment
191 A, a representative immunoblot of whole-cell lysates for wild-type BCRP and the mutants K452A, K453D, H457A, R465A, and K473A. Login to comment
207 After normalization to the BCRP levels of whole-cell lysates, statistically significant differences in efflux activities for all three substrates were noticed for K452A, K453D, H457A, and R465A compared with wild-type protein. Login to comment
209 Thus, the efflux activities of K452A and H457A were significantly increased 2to 6-fold, whereas the activities of K453D and R465A were decreased by 40 to 60%, depending on substrate (Table 1). Login to comment
220 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. Login to comment
221 Thus, the relative levels of resistance of K452A and H457A to MX were increased approximately 2to 3-fold compared with wild-type protein, whereas those of K453D and R465A to MX were decreased by 30 to 70%. Login to comment
222 Likewise, the relative levels of resistance of H457A to SN-38 were increased approximately 3-fold, whereas those of K453D and R465A to SN-38 were decreased by 50 to 60%. Login to comment
232 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. Login to comment
234 After normalization to the BCRP levels, the Vmax values of K453D and R465A were approximately 60% lower than that of wild-type BCRP, whereas the Vmax values of other mutants did not change. Login to comment
235 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. Login to comment
241 Selected areas of HEK cells expressing wild-type BCRP and the mutants K452A, K453D, H457A, R465A, and K473A are shown. Login to comment
244 In contrast, the Vmax/Km values of K453D and R465A were decreased by approximately 40 to 70%. Login to comment
263 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. Login to comment
264 However, the addition of prazosin differentially increased the binding of 5D3 to wild-type BCRP, K452A, K453D, H457A, and R465A in a concentration-dependent manner (Fig. 6), suggesting that the binding equilibrium between prazosin and BCRP could be monitored by measuring the binding of 5D3 to the transporter. Login to comment
265 Thus, the apparent dissociation constants of the prazosin complex with wild-type or mutant BCRP were estimated to be 5.3 Ϯ 1.1, 14.7 Ϯ 2.3, 3.1 Ϯ 0.4, 20.1 Ϯ 4.0, and 6.7 Ϯ 1.8 ␮M for wild-type BCRP, K452A, K453D, H457A, and R465A, respectively. Login to comment
289 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). Login to comment
297 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. Login to comment
317 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. Login to comment
319 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 (‚). Login to comment
329 In contrast, substitutions of Lys453 and Arg465 with Asp (K453D) or Ala (R465A) caused a nonselective global reduction in BCRP activity (Tables 1 and 2). Login to comment
363 K452A and H457A were associated with a 50 to 70% decrease in Km and a 2to 5-fold increase in Vmax/Km for ATP hydrolysis, whereas the Vmax/Km values of K453D and R465A were decreased by 40 to 70%, and the Km values of K453D and R465A did not change much (Table 3). Login to comment
364 This suggests that ATP binding affinity and/or the efficiency of ATP hydrolysis are increased for K452A and H457A, but decreased for K453D and R465A, thus affecting BCRP activity accordingly. Login to comment
375 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). Login to comment

[hide] Determinants of the activity and substrate recogni... Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18. Szafraniec MJ, Szczygiel M, Urbanska K, Fiedor L
Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).
Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18., [PMID:25036722]

Abstract [show]
The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.

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No. Sentence Comment
209 Position Type of mutation Effect on the transporter References NBD Lys 86 Met (i) No stimulation of the ATPase activity by prazosin; (ii) no influence on the transport of mitoxantrone Henriksen et al. (2005b) Glu 126 stop, Phe 208 Ser, Ser 248 Phe, Glu 334 stop Inability to transport hematoporphyrin Tamura et al. (2006) Glu 211 Gln Complete abolishment of the ATPase activity and methotrexate transport Hou et al. (2009) Pro 392 Ala Significant reduction in the efflux activity of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Ni et al. (2011) TM1 Gly 406 Ala Gly 410 Ala No influence on the activity of the transporter Polgar et al. (2004) Gly 406 Leu Gly 410 Leu (i) Loss of the ability to transport rhodamine123; (ii) impaired transport of mitoxantrone, Pheide and BODIPY-prazosin Polgar et al. (2004) Extracellular loop 1 Phe 431 Leu (i) Loss of the ability to transport methotrexate; (ii) 10% level of hematoporphyrin transport compared to the WT protein Tamura et al. (2006) Ser 441 Asn Inability to transport hematoporphyrin Tamura et al. (2006) Ser 441 Asn Loss of the ability to transport methotrexate Tamura et al. (2006) TM2 Lys 452 Ala His 457 Ala Increase in transport of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Cai et al. (2010) Lys 453 Ala Arg 465 Ala Decrease in transport of mitoxantrone, BODIPY-prazosin, Hoechst 33342, doxorubicin, SN-38 and rhodamine 123 Cai et al. (2010) TM3 Arg 482 Gly Arg 482 Thr (i) No change in the inhibitory activity of lapatinib; (ii) about two times greater inhibition by ritonavir, saquinavir and nalfinavir than in the WT variant; (iii) gaining the ability to transport rhodamine123 and doxorubicin; (iv) no influence on the transport of mitoxantrone; (v) loss of the ability to transport methotrexate Dai et al. (2008), Gupta et al. (2004), Honjo et al. (2001), Mitomo et al. (2003) Arg 482 Thr (i) Lower IC 50 of cyclosporine A for mutant than for WT variant; (ii) lower elacridar inhibition potency Xia et al. (2007) Arg 482 Lys Complete loss of transport activity Ejendal et al. (2006) Phe 489 Leu Impaired transport of porphyrins, no transport of methotrexate Tamura et al. (2006) Extracellular loop 3 Asn 590 Tyr Over twice reduced transport of mitoxantrone, topotecan, daunorubicin and rhodamine 123 Vethanayagam et al. (2005) Cys 592 Ala/Cys 608 Ala (i) Transport of mitoxantrone almost unchanged; (ii) transport of BODIPY-prazosin significantly impaired Henriksen et al. (2005a) Extracellular loop 3 Cys 603 Ser Cys 592 Ser/Cys 608 Ser Cys 592 Ser/Cys 603 Ser/Cys 608 Ser Diminished susceptibility to the inhibitory activity of fumitremorgin C Shigeta et al. (2010) Cys-less Arg 482 Gly-BCRP Complete loss of the ability to efflux mitoxantrone Liu et al. (2008b) The positions of the amino acid residues refer to the topological model of BCRP proposed by Wang et al. (2009). Login to comment