ABCMdb

PMID: 20203106

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., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCG2 p.Lys473Ala Lys452 , Lys453 , His457 , Arg465 , and Lys473 were replaced with Ala or Asp. Login to comment 3 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 7 ABCG2 p.Lys473Ala The transport activities and drug-resistance profiles of K473A were not changed. Login to comment 8 ABCG2 p.His457Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp 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 32 ABCG2 p.Arg482Thr Rhodamine123 [Rho123; 2Ј-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-5Ј-bi-1h-benzimidazole] and anthracyclines such as doxorubicin (Dox) and daunorubicin are not substrates of wild-type BCRP, but can be efficiently transported by the mutants with Gly or Thr substitution of Arg482 (Honjo et al., 2001; Miwa et al., 2003; Ozvegy-Laczka et al., 2005). Login to comment 76 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala 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 178 ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp Because substitution of Lys453 with Asp showed substantially higher levels of expression than substitution with Ala in all the clones obtained in this study, we examined substitution of Lys453 with Asp that may affect BCRP activity if the positive charge of Lys453 is functionally essential. Login to comment 183 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 208 ABCG2 p.Lys473Ala Efflux activities of K473A were not significantly changed. Login to comment 209 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp 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 210 ABCG2 p.His457Ala Notably, the activities of H457A for BODIPY-prazosin and Hoechst33342 were increased 3-to 6-fold, but its activity for MX was increased only 1.7-fold. Login to comment 220 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys453Asp 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 223 ABCG2 p.Lys452Ala However, the relative levels of resistance of K452A to SN-38 did not change much. Login to comment 232 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Arg465Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp Selected areas of HEK cells expressing wild-type BCRP and the mutants K452A, K453D, H457A, R465A, and K473A are shown. Login to comment 244 ABCG2 p.Arg465Ala ABCG2 p.Lys453Asp In contrast, the Vmax/Km values of K453D and R465A were decreased by approximately 40 to 70%. Login to comment 245 ABCG2 p.Lys473Ala The Vmax/Km value of K473A was comparable with that of wild-type protein. Login to comment 263 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 266 ABCG2 p.Lys473Ala In contrast, prazosin only slightly decreased, rather than increased, the binding of 5D3 to K473A at low concentrations. Login to comment 268 ABCG2 p.Lys473Ala ABCG2 p.His457Ala Limited Trypsin Digestion of Wild-Type BCRP and the Mutants H457A and K473A. Login to comment 270 ABCG2 p.Lys473Ala ABCG2 p.His457Ala 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 309 ABCG2 p.His457Ala ABCG2 p.His457Ala ABCG2 p.Lys452Ala ABCG2 p.Lys452Ala Notably, replacing Lys452 or His457 with Ala (K452A or H457A) markedly increased the efflux of MX, BODIPY-prazosin, and Hoechst33342 (Table 1). Login to comment 310 ABCG2 p.His457Ala ABCG2 p.Lys452Ala Likewise, K452A and H457A conferred significantly increased resistance to MX and SN-38 compared with wild-type BCRP (Table 2). Login to comment 312 ABCG2 p.Arg482Gly For example, R482G and TABLE 3 Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP ATP hydrolysis activities of plasma membrane preparations from HEK293 cells expressing wild-type and mutant BCRP were determined as described under Materials and Methods. Login to comment 317 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala 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 ABCG2 p.Lys473Ala ABCG2 p.His457Ala Trypsin digestion of wild-type BCRP, H457A, and K473A. Login to comment 322 ABCG2 p.Lys473Ala ABCG2 p.His457Ala 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 326 ABCG2 p.Arg482Thr R482T exhibited markedly increased resistance to MX (Miwa et al., 2003; Robey et al., 2003). Login to comment 328 ABCG2 p.His457Ala ABCG2 p.Lys452Ala It is worth noting that K452A and H457A are not selected during evolution. Login to comment 329 ABCG2 p.Arg465Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp ABCG2 p.Arg465Asp 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 331 ABCG2 p.Lys473Ala Conversion of Lys473 to Ala had no significant effect on BCRP activity. Login to comment 339 ABCG2 p.Lys453Asp Substitution of Lys453 with Asp would abolish the salt bridge and affect the shape of substrate binding sites and the conformation of BCRP, leading to an overall loss of transport activity. Login to comment 363 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp ABCG2 p.Lys453Asp 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 ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 367 ABCG2 p.Arg482Thr More importantly, the Km and Vmax/Km values for basal ATPase activity of R482T with a similar gain of function have also been shown to be decreased and increased, respectively (Pozza et al., 2006). Login to comment 375 ABCG2 p.Lys473Ala ABCG2 p.His457Ala ABCG2 p.Arg465Ala ABCG2 p.Lys452Ala ABCG2 p.Lys453Asp 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 376 ABCG2 p.His457Ala ABCG2 p.Lys452Ala Intriguingly, the apparent dissociation constant of the prazosin complex with K452A or H457A was increased approximately 3-to 4-fold compared with wild-type BCRP, suggesting that the association rate of prazosin to BCRP may be decreased and/or prazosin could dissociate from the BCRP-prazosin complex more readily. Login to comment 377 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.His457Ala ABCG2 p.Lys452Ala Given the nature of gain of function associated with K452A and H457A, a similar observation was reported in which R482T was shown to be much less intensively photolabeled by a photoactive analog of Rho123 than wild-type BCRP (Alqawi et al., 2004), indicating the binding affinity of the photoactive substrate to R482T was decreased even though R482T can effectively transport Rho123, but wild-type BCRP cannot. Login to comment 379 ABCG2 p.Lys473Ala 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