ABCMdb

ABCG2 p.Lys473Ala

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Publications

PMID: 20203106 [PubMed] Cai X et al: "Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport."

No. Sentence Comment

2 Lys452 , Lys453 , His457 , Arg465 , and Lys473 were replaced with Ala or Asp. Login to comment
7 The transport activities and drug-resistance profiles of K473A were not changed. 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
177 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. 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
208 Efflux activities of K473A were not significantly changed. 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
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
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
245 The Vmax/Km value of K473A was comparable with that of wild-type protein. 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
266 In contrast, prazosin only slightly decreased, rather than increased, the binding of 5D3 to K473A at low concentrations. Login to comment
268 Limited Trypsin Digestion of Wild-Type BCRP and the Mutants H457A and K473A. Login to comment
270 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). Login to comment
273 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). 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
320 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. Login to comment
321 Trypsin digestion of wild-type BCRP, H457A, and K473A. Login to comment
322 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. Login to comment
331 Conversion of Lys473 to Ala had no significant effect on BCRP activity. 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
379 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. Login to comment

PMID: 20812902 [PubMed] Ni Z et al: "Structure and function of the human breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

314 Such mutants include K473A and H630X, suggesting that these residues are likely not critical for expression and function of BCRP. Login to comment

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

109 We made individual substitutions of each amino acid to alanine: M131A, S195A, K453A, K473A, P485A, M549A, W564A and I573A. Login to comment
153 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). Login to comment