ABCMdb

PMID: 19691360

Hou YX, Li CZ, Palaniyandi K, Magtibay PM, Homolya L, Sarkadi B, Chang XB
Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport.
Biochemistry. 2009 Sep 29;48(38):9122-31., 2009-09-29 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCG2 p.Glu211Gln Substitution of the putative catalytic residue E211 with a nonacidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport. Login to comment 4 ABCG2 p.Glu211Gln To address this question, we have made an YFP/ABCG2 fusion protein and expressed this 99 kDa fusion protein alone or along with the 70 kDa E211Q-mutated ABCG2 in BHK cells. Login to comment 6 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Interestingly, membrane vesicles prepared from BHK cells expressing both YFP/ABCG2 and E211Q-mutated ABCG2 (with a ratio of 1:1) form homodimers and heterodimer and exert 55% of wt ABCG2 ATPase activity that can be further enhanced by anticancer drugs, suggesting that the wt NBD in the heterodimer of YFP/ABCG2 and E211Q may be able to hydrolyze ATP. Login to comment 7 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Furthermore, themembrane vesicles containing both YFP/ABCG2 and E211Q exert ~79% of wt ABCG2-mediated methotrexate transport activity, implying that the heterodimer harboring YFP/ABCG2 and E211Q may be able to transport the anticancer drug methotrexate across the biological membranes. Login to comment 23 ABCG2 p.Glu211Gln Interestingly, E211Q mutation (in homodimer) completely abolished its ATPase activity and ATP-dependent anticancer drug transport, whereas coexpression of this mutant † This work was supported by a grant from the National Cancer Institute (CA89078 to X.C.). Login to comment 39 ABCG2 p.Glu211Gln The putative catalytic residue E211 was mutated to Q in pNUT/ABCG2, named as pNUT/ABCG2/E211Q. Login to comment 40 ABCG2 p.Glu211Gln In order to distinguish the wt ABCG2 from the E211Q-mutated protein, the full-length yellow fluorescent protein (YFP) cDNA (238 amino acids) with the SGLRSRAAANT (11 amino acids) linker (45), amplified by PCR from the plasmid DNA containing YFP cDNA (46), was inserted into the N-terminus of pNUT/ABCG2, named as pNUT/YFP/ABCG2. Login to comment 77 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln In order to test whether substitution of the ABCG2 putative catalytic base E211 with a glutamine residue (E211Q) is able to transport MTX across biological membranes, membrane vesicles have been prepared from the BHK cells expressing either wt or E211Q-mutated ABCG2. Login to comment 78 ABCG2 p.Glu211Gln The results in Figure 1A indicate that the amount of wt ABCG2 is similar to that of E211Q, whereas there is no detectable amount of ABCG2 in membrane vesicles prepared from the parental or cystic fibrosis transmembrane-conductance regulator (CFTR or ABCC7) cDNA-transfected BHK cells. Login to comment 80 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Although the amount of E211Q-mutated ABCG2 is similar to that of wt ABCG2, E211Q mutated ABCG2, similar to CFTR and BHK membrane vesicles, was unable to transport MTX across the biological membranes (Figure 1B), suggesting that substitution of the putative catalytic base of ABCG2 with a nonacidic glutamine residue may completely abolish its ATPase activity. Login to comment 84 ABCG2 p.Glu211Gln The Heterodimer of YFP/ABCG2 and E211Q May Be Able To Transport MTX across the Biological Membranes. Login to comment 85 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Since E211Q-mutated ABCG2 completely abrogated its ATP-dependent MTX transport activity (Figure 1B), we had asked a question of whether the heterodimer of wt and E211Q-mutated ABCG2 was able to transport MTX across the biological membranes or not. Login to comment 86 ABCG2 p.Glu211Gln However, because the wt ABCG2 and the E211Q-mutated ABCG2 run to the same position in SDS-PAGE (as shown in Figure 1A), there was no way to distinguish these two proteins if they were coexpressed in BHK cells. Login to comment 89 ABCG2 p.Glu211Gln (A) Expression of wt and E211Q-mutated ABCG2 in BHK cells. Login to comment 92 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln (B) E211Q-mutated ABCG2 isunabletotransport MTX intothe membranevesicles.The dATP(4 mM) dependent MTX transport by ABCG2, E211Q, BHK, or CFTR membrane vesicles was performed in triplicate according to the method described in Experimental Procedures. Login to comment 100 ABCG2 p.Glu211Gln The complex-glycosylated YFP/ABCG2 has an apparent molecular mass of ~99 ( 3 kDa (Figure 2B, n = 16), which is significantly larger than its unmodified wt or E211Q-mutated ABCG2, with an apparent molecular mass of ~70 ( 2 kDa (Figure 2B, n = 24). Login to comment 101 ABCG2 p.Glu211Gln Due to the mobility difference between YFP/ABCG2 and E211Q-mutated ABCG2, these two proteins coexpressed in BHK cells can be separated very well (Figure 2B). Login to comment 102 ABCG2 p.Glu211Gln Furthermore, the ratio between the complex-glycosylated YFP/ABCG2 and E211Q, probed with the same ABCG2-specific mAb BXP-21, can be accurately determined. Login to comment 103 ABCG2 p.Glu211Gln Thus, YFP/ABCG2 was coexpressed with E211Q (with plasmid DNA ratios of 2:8, 4:6, 5:5, 6:4, or 8:2) in BHK cells. Login to comment 105 ABCG2 p.Glu211Gln Colonies derived from the transfections with ratios of 4:6, 5:5, or 6:4 yielded similar amounts of YFP/ABCG2 and E211Q. Login to comment 106 ABCG2 p.Glu211Gln Membrane vesicles were prepared from the colonies with similar amounts of YFP/ABCG2 and E211Q and analyzed by Western blots. Login to comment 107 ABCG2 p.Glu211Gln Membrane vesicles containing the same amounts of YFP/ ABCG2 and E211Q (with a ratio of 1:1 as shown in Figure 2B) were used to do further analysis. Login to comment 108 ABCG2 p.Glu211Gln FIGURE 2: Heterodimer of YFP/ABCG2 and E211Q-mutated ABCG2 may be able to transport MTX across the biological membranes. Login to comment 112 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Membrane vesicles (1 μg) containing wt ABCG2, YFP/ABCG2, E211Q or YFP/ABCG2 + E211Q were subjected to SDS-PAGE (7%) and probed with ABCG2 mAb BXP-21. Login to comment 114 ABCG2 p.Glu211Gln (C) Coexpression of YFP/ABCG2 and E211Q in BHK cells forms homodimers and heterodimer. Login to comment 115 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Membrane vesicles containing wt ABCG2 (0.5 μg), YFP/ABCG2 (0.5 μg), E211Q (0.5 μg), or YFP/ABCG2 + E211Q (1 μg) were subjected to SDS-PAGE (4-10% gradient gel) in the absence of reducing agent DTT and probed with ABCG2 mAb BXP-21. Login to comment 118 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Mm, Hmd, and Htd on the right indicate the monomers of ABCG2, E211Q, or YFP/ABCG2, the homodimers of ABCG2, E211Q, or YFP/ABCG2, and the heterodimer of YFP/ABCG2 and E211Q. Login to comment 119 ABCG2 p.Glu211Gln (D) E211Q-mutated ABCG2 is unable to hydrolyze ATP. Login to comment 120 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The ATPase assay (in triplicate) was performed according to the method described in Experimental Procedures by using 2 μg of the same membrane vesicles shown in panels B and C (1.8862 μg of wt ABCG2 + 0.1138 μg of BHK; 1.5145 μg of YFP/ABCG2 + 0.4855 μg of BHK; 2 μg of E211Q; and 0.7918 μg of YFP/ABCG2 + E211Q + 1.2082 μg of BHK) and 4 mM ATP at 37 °C for 30 min. After subtraction of the amount of inorganic phosphate generated in the presence of 2 μg of BHK membrane vesicles, the velocity of the ATPase activity was calculated and compared to that of ABCG2 (n = 5). Login to comment 121 ABCG2 p.Glu211Gln (E) The heterodimer of YFP/ABCG2 and E211Q may be able to transport MTX across the biological membranes. Login to comment 122 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The MTX uptake assays were carried out, according to the method described in Experimental Procedures, in a 30 μL solution (in triplicate) containing 3 μg of membrane vesicles (2.829 μg of ABCG2 + 0.171 μg of BHK; 2.272 μg of YFP/ABCG2 + 0.728 μg of BHK; 3 μg of E211Q; and 1.188 μg of YFP/ABCG2 + E211Q + 1.812 μg of BHK), 2 mM MTX, 10 mM DTT, and 4 mM dATP at 37 °C for 7.5 min. After subtraction of the amount of radioactivity bound to the nitrocellulose membrane in the presence of 4 mM AMP from the same sample in the presence of 4 mM dATP, the velocity of the dATP-dependent MTX transport was calculated (n = 4). Login to comment 123 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln When the membrane vesicles were separated in SDS-PAGE in the absence of reducing agent DTT, wt ABCG2 or E211Q monomer (70 ( 2 kDa) and homodimer (149 ( 0 kDa) and YFP/ ABCG2 monomer (99 ( 3 kDa) and homodimer (220 ( 1 kDa) and heterodimer of YFP/ABCG2 and E211Q (188 ( 0 kDa) were clearly detected (Figure 2C). Login to comment 124 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Interestingly, in the 4-10% gradient gel (Figure 2C), the ratio of E211Q and YFP/ABCG2 is approximately 1:1.1 ( 0.2, implying that similar amounts of E211Q and YFP/ABCG2 form either homodimer or heterodimer. Login to comment 125 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The ratios between the homodimers and heterodimer further confirm this conclusion, i.e., homodimer of YFP/ABCG2 (30.0 ( 3.2%, n = 25), homodimer of E211Q (28.2 ( 3.8%, n = 25), and the heterodimer of YFP/ABCG2 and E211Q (41.8 ( 3.6%, n = 25). Login to comment 126 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Thus, regardless of whether the intermolecular disulfide bond is formed in vivo or in vitro, the heterodimer of YFP/ABCG2 and E211Q does exist in the plasma membranes of the BHK cells cotransfected with YFP/ABCG2 and E211Q-mutated ABCG2. Login to comment 130 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The ratio between YFP/ABCG2 and E211Q is ~1.0375 ( 0.0303 (n = 4), indicating that the amount of YFP/ ABCG2 protein in the membrane vesicles containing both YFP/ ABCG2 and E211Q is not significantly different from that of E211Q-mutated ABCG2. Login to comment 131 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The ratios between different samples and wt ABCG2 are as follows: 2.3821 ( 0.3762 (YFP/ABCG2 þ E211Q, including both YFP/ABCG2 and E211Q bands, versus wt ABCG2, n = 4); 0.9431 ( 0.1758 (E211Q versus wt ABCG2, n = 4); 1.2454 ( 0.1878 (YFP/ABCG2 versus wt ABCG2, n = 4). Login to comment 132 ABCG2 p.Glu211Gln The results in Figure 2D indicate that wt ABCG2 is able to hydrolyze ATP, with a velocity of ~ 40 nmol mg-1 min-1 , whereas YFP/ABCG2, after adjusted with BHK membrane vesicles to have the same amount of ABCG2 protein, is moder- atelymoreactive thanthat of wt ABCG2.However,E211Q alone is unable to hydrolyze ATP, indicating that substitution of the putative catalytic residue E211 with a glutamine residue completely abolished its ATPase activity. Login to comment 133 ABCG2 p.Glu211Gln Coexpression of YFP/ ABCG2 with E211Q yielded ~55% of wt ABCG2 ATPase activity (Figure 2D), making it difficult to make any conclusion from this result. Login to comment 134 ABCG2 p.Lys86Met ABCG2 p.Glu211Gln However, in considering the results published by Henriksen et al., i.e., coexpression of wt ABCG2 with K86M-mutated ABCG2 exerted ~50% of wt ABCG2 ATPase activity (54), the above result might be interpreted as that one of the two ATPs bound to the heterodimer of YFP/ABCG2 and E211Q could be hydrolyzed. Login to comment 137 ABCG2 p.Glu211Gln In contrast, E211Q alone is unable to transport MTX across the biological membranes (Figure2E). Login to comment 138 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln However,coexpression of YFP/ABCG2 with E211Q (with a ratio of 1:1), after adjusted with BHK membrane vesicles to have similar amount of ABCG2 in YFP/ ABCG2 þ E211Q as in wt ABCG2, yielded approximately 79% of wt ABCG2 transport activity (Figure 2E and Table 1), suggesting that the heterodimer of YFP/ABCG2 and E211Q may be able to transport MTX into the membrane vesicles. Login to comment 139 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Heterodimer Formation of YFP/ABCG2 and E211Q Altered the Kinetic Parameters of Nucleotide-Dependent MTX Transport. If the heterodimer of YFP/ABCG2 and E211Q is able to transport MTX into the membrane vesicles, we expect that the kinetic parameters of nucleotide-dependent MTX transport may be changed. Login to comment 143 ABCG2 p.Glu211Gln In contrast, the ATP- (Figure 3C) and dATP-dependent (Figure 3D) MTX transport by membrane vesicles containing both YFP/ABCG2 and E211Q exerted two (mixed) Michaelis-Menten curves: one yields lower Km and Vmax values and the other higher Km and Vmax values (due tooverlap between these two curves, the Km and Vmax values cannot be accurately determined). Login to comment 144 ABCG2 p.Glu211Gln The results were interpreted as that the MTX transported into membrane vesicles at lower concentrations of nucleotides was catalyzed by the homodimer of YFP/ABCG2, whereas the one transported into membrane vesicles at higher concentrations of nucleotides was catalyzed by both the homodimer of YFP/ABCG2 and heterodimer of YFP/ABCG2 and E211Q. Login to comment 147 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The results in Figure 4 indicated that the basal ATPase activity (~ 40 nmol mg-1 min-1 ) of membrane vesicles derived from the BHK cell is the same as the one derived from the BHK/E211Q cell, indicating that E211Q protein does not have ability to hydrolyze ATP. These results also indicated that EGTA, ouabain, and sodium azide did not completely inhibit all of the ATPases in BHK membrane vesicles. Login to comment 148 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln The basal ATPase activities of membrane vesicles containing wt ABCG2 (~80 nmol mg-1 min-1 without subtracting the ATPase activity contributed by BHK membrane proteins), YFP/ABCG2 (~105 nmol mg-1 min-1 ), or YFP/ABCG2 þ E211Q (~76 nmol mg-1 min-1 ) were significantly higher than that of BHK or E211Q membrane vesicles, indicating that wt ABCG2, YFP/ABCG2, or YFP/ABCG2 þ E211Q can hydrolyze ATP in the absence of their substrate. Login to comment 149 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Furthermore, the basal ATPase activities of either BHK membrane proteins or E211Q cannot be enhanced by anticancer drugs, such as daunomycin (Figure 4A), methotrexate (Figure 4B), or tamoxifen (Figure 4C), whereas the ATPase activities wt ABCG2, YFP/ABCG2, or YFP/ABCG2 þ E211Q Table 1: Relative MTX Transport Activitya sample MTX transport (%) ABCG2 100.0 ( 0.0 YFP/ABCG2 101.2 ( 2.4 E211Q 3.7 ( 3.3 YFP/ABCG2 + E211Q 79.4 ( 11.2 a The data were derived from Figure 2E (n = 4). Login to comment 152 ABCG2 p.Glu211Gln Substitution of the putative catalytic E211 residue with a noncharged amino acid glutamine (E211Q) completely abolished its ATPase activity (Figures 2 and 4), suggesting that the acidic amino acidE211 plays a veryimportant role in ATP hydrolysis. Login to comment 153 ABCG2 p.Glu211Gln E211Q mutation also completely abolished the dATP-dependent MTX transport (Figures 1 and 2), indicating that ATP hydrolysis is required for facilitating the anticancer drug MTX across the biological membranes. These results are consistent with the corresponding mutations in ABCB1 (57), in ABCC1 (E1455 mutations in NBD2) (58, 59), and in bacterial ABC transporters MJ0796 (18) and HisP (27). Login to comment 155 ABCG2 p.Glu211Gln Since ABCG2 forms homodimer in vivo, the E211Q mutation actually mutated both putative catalytic bases in the two NBDs of the homodimer of this protein. Login to comment 161 ABCG2 p.Glu211Gln (C) ATP-dependent MTX transport by YFP/ABCG2 + E211Q. Login to comment 162 ABCG2 p.Glu211Gln (D) dATP-dependent MTX transport by YFP/ABCG2 + E211Q. Login to comment 167 ABCG2 p.Glu211Gln ular mass of 99 kDa) so that it can be clearly distinguished from the E211Q-mutated ABCG2 (with an apparent molecular mass of 70 kDa). Login to comment 172 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Thus, if the YFP/ABCG2 fusion protein can form heterodimer with E211Q-mutated ABCG2, this heterodimer may be used to test whether the ATP bound in the NBD3 ATP3 ATP3 NBD-E211Q sandwich structure can be hydrolyzed or not. Login to comment 173 ABCG2 p.Glu211Gln Indeed, when YFP/ABCG2 was coexpressed with E211Q-mutated ABCG2 in BHK cells, approximately 42% of them form heterodimer in the BHK plasma membranes (Figure 2C). Login to comment 174 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Although E211Q mutation completely abolished its ATPase activity (Figures 2D and 4), coexpression of YFP/ABCG2 with E211Q-mutated ABCG2 exerted ~55% of wt ABCG2 ATPase activity (Figure 2D). Login to comment 175 ABCG2 p.Lys86Met This is consistent with Henriksen`s result that coexpression of wt ABCG2 with K86M-mutated ABCG2 exerted ~50% of wt ABCG2 ATPase activity (54). Login to comment 176 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln This ATPase activity, regardless ofwhether itiscontributed by wtNBD fromhomodimer of YFP/ ABCG2 or heterodimer containing YFP/ABCG2 and E211Q-mutated ABCG2, can be further stimulated by anticancer drugs, such as daunomycin (Figure 4A), MTX (Figure 4B), or tamoxifen (Figure 4C), implying that one of the two ATPs bound in the NBD3ATP3ATP3NBD-E211Q sandwich structure could be hydrolyzed. Login to comment 177 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Of course, this conclusion should be further confirmed by using covalently linked dimers (64), such as wt ABCG2-wt ABCG2, E211Q-E211Q, and wt ABCG2-E211Q (which is our ongoing project). Login to comment 178 ABCG2 p.Glu211Gln The above conclusion implies that one of the two ATPs bound in the NBD3ATP3ATP3NBD-E211Q sandwich structure could be hydrolyzed by wt NBD. Login to comment 179 ABCG2 p.Glu211Gln If that is the case, the heterodimer of YFP/ABCG2 and E211Q may be used to address the question of whether one ATP hydrolysis or two ATP hydrolyses in the NBD3ATP3ATP3NBD sandwich structure is/are required for the ATP-dependent solute transport by ABCG2. Login to comment 184 ABCG2 p.Lys86Met ABCG2 p.Asp210Asn ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln Furthermore, membrane vesicles prepared from BHK/CFTR (Figure 1) and BHK/E211Q (Figures 1 and 2), which were generated under the same selection procedures as YFP/ABCG2 þ E211Q, were unable to transport MTX across the biological membranes. These results suggest that the heterodimer containing YFP/ ABCG2 and E211Q-mutated ABCG2 is able to transport the bound MTX into the membrane vesicles, strikingly contrasting the conclusions derived from K86M- or D210N-mutated ABCG2 (11, 54, 64). Login to comment 186 ABCG2 p.Glu211Gln Kinetic analyses of the membrane vesicles containing both YFP/ABCG2 and E211Q-mutated ABCG2 (Figure 3C,D) indicate that there might be two populations of membrane vesicles: one population has lower Km and Vmax values and the other higher Km and Vmax values. Login to comment 189 ABCG2 p.Glu211Gln the conclusion made by McDevitt et al. (65), i.e., ATP binding to NBD of ABCG2 induces conformational changes that result in shifting the bound anticancer drug from the high- to low-affinity site, we interpreted these results as that ATP binding to the heterodimer of E211Q and YFP/ABCG2 might induce proper conformational changes that brought the bound MTX from the high- to low-affinity site. Login to comment 190 ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln ABCG2 p.Glu211Gln One ATP hydrolysis in the NBD3ATP3 ATP3NBD-E211Q sandwich structure, catalyzed by the wt NBD, may facilitate the release of both nucleotides bound in thissandwich structureandbringthe moleculeback toits original conformation so that the protein can start a new cycle of ATP-dependent solute transport. If this is the case, the higher Km value of a population, possibly the heterodimer of YFP/ABCG2 and E211Q, implies that higher concentration of nucleotide is required to form the NBD3ATP3ATP3NBD-E211Q sandwich structure. Login to comment 191 ABCG2 p.Lys86Met ABCG2 p.Lys86Met ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn ABCG2 p.Glu211Gln If K86M- or D210N-mutated ABCG2 had higher effects on ATP binding than E211Q, much higher concentration of ATP may be required to form the NBD3ATP3ATP3NBD- K86M or NBD3ATP3ATP3NBD-D210N sandwich structure. Login to comment