ABCMdb

PMID: 21854076

Ni Z, Bikadi Z, Shuster DL, Zhao C, Rosenberg MF, Mao Q
Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity.
Biochemistry. 2011 Sep 20;50(37):8057-66. Epub 2011 Aug 26., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin. Login to comment 7 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP. Login to comment 9 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner. Login to comment 46 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ABCG2 p.Pro480Ala ABCG2 p.Pro574Ala ABCG2 p.Pro501Ala ABCG2 p.Pro623Ala Full-length wild-type human BCRP cDNA in pcDNA3.1 was subcloned into pcDNA5/ FRT.15 The pcDNA5/FRT plasmid containing full-length human BCRP cDNA was used as a template to generate all the BCRP mutants using PCR mutagenesis as previously described.15 The following forward primers were used for mutagenesis: P392A (5'-AAC TTG CTG GGT AAT GCC CAG GCC TCT ATA GCT C-3'), P480A (5'-CTG TTA TCT GAT TTA TTA GCC ATG AGG ATG TTA CCA AG-3'), P485A (5'-CCC ATG AGG ATG TTA GCA AGT ATT ATA TTT ACC TGT ATA G-3'), P501A (5'-CAT GTT AGG ATT GAA GGC AAA GGC AGA TGC CTT C-3'), P574A (5'-CAG TAC TTC AGC ATT GCA CGA TAT GGA TTT ACG-3'), and P623A (5'-GGC ATC GAT CTC TCA GCC TGG GGC TTG TGG AAG AAT-3'). Login to comment 79 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala (D) Protein expression levels of P392A and P485A relative to that of wild-type BCRP (100%) in the plasma membrane samples. Login to comment 104 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ABCG2 p.Pro480Ala ABCG2 p.Pro574Ala ABCG2 p.Pro501Ala ABCG2 p.Pro623Ala The estimated relative cell surface expression levels of wild-type and mutant BCRP based on the differences in median fluorescence between 5D3 and IgG2b control incubations were 100 ± 22, 108 ± 4, 89 ± 1, 139 ± 32, 75 ± 16, 76 ± 9, and 114 ± 0.7% for three independent determinations for wild-type BCRP, P392A, P480A, P485A, P501A, P574A, and P623A, respectively. Login to comment 106 ABCG2 p.Pro485Ala The 5D3 fluorescence associated with P485A was increased by 39% compared to that of wild-type BCRP, but the difference was not statistically significant (p > 0.05 by a Figure 3. Login to comment 124 ABCG2 p.Pro392Ala As shown in Table 1, only P392A exhibited a significantly lower resistance to MX compared to that of wild- Figure 4. Login to comment 129 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ABCG2 p.Pro480Ala ABCG2 p.Pro574Ala ABCG2 p.Pro501Ala ABCG2 p.Pro623Ala The pcDNA5 vector control, wild-type BCRP, P392A, P480A, P485A, P501A, P574A, and P623A are indicated by P, WT, 392, 480, 485, 501, 574, and 623, respectively. Login to comment 132 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ABCG2 p.Pro480Ala ABCG2 p.Pro574Ala ABCG2 p.Pro501Ala ABCG2 p.Pro623Ala Drug Resistance Profile of Wild-Type and Mutant BCRPa MX SN-38 Dox Rho123 IC50 (nM) RR IC50 (nM) RR IC50 (nM) RR IC50 (nM) RR pcDNA5/FRT 5.6 ± 0.8 - 1.7 ± 0.1 - 33.1 ± 2.2 - 4218 ± 400.0 - wild-type BCRP 51.5 ± 8.5 9.2 30.3 ± 2.0 17.8 40.9 ± 6.7 1.2 4446 ± 261.5 1.1 P392A 24.6 ± 3.8 4.4* 13.1 ± 1.0 7.7* 34.8 ± 1.7 1.1 5326 ± 414.1 1.3 P480A 69.4 ± 10.5 12.4 37.8 ± 1.5 22.2 29.0 ± 4.0 0.9 5107 ± 272.7 1.2 P485A 38.5 ± 7.0 6.9 15.9 ± 2.6 9.4* 41.8 ± 5.3 1.3 3237 ± 199.5 0.8 P501A 40.9 ± 14.6 7.3 27.5 ± 2.4 16.2 23.2 ± 3.8 0.7 4480 ± 816.4 1.1 P574A 49.6 ± 12.5 8.9 32.5 ± 2.6 19.1 29.6 ± 3.5 0.9 5320 ± 419.8 1.3 P623A 68.4 ± 14.4 12.2 36.8 ± 2.8 21.6 37.0 ± 8.8 1.1 3302 ± 388.1 0.8 a The relative resistance (RR) values represent the relative levels of resistance of the mutants compared to that of wild-type BCRP and were calculated by dividing the IC50 values of wild-type and mutant BCRP by the IC50 values of the vector control. Login to comment 137 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala ABCG2 p.Pro485Ala On the other hand, both P392A and P485A showed significantly decreased resistance to SN-38 compared to that of the wild type, thus providing additional evidence that alanine substitution of Pro485 changed the substrate specificity of BCRP. Login to comment 142 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala To examine if the changes in efflux activity of mutant BCRP are caused by alterations in its ability to hydrolyze ATP, we measured vanadate-sensitive ATPase activities of wild-type BCRP, P392A, and P485A in plasma membrane preparations. Login to comment 143 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala The protein expression levels of wild-type BCRP, P392A, and P485A in plasma membranes were determined by immunoblotting using mAb BXP-21 and found to be comparable after normalization to the internal plasma membrane control Na+ / K+ -ATPase (Figure 2C,D). Login to comment 145 ABCG2 p.Pro392Ala P392A exhibited ATPase activities similar to that of wild-type BCRP. Login to comment 146 ABCG2 p.Pro485Ala P485A exhibited a modest decrease in ATPase activity compared to that of wild-type BCRP; however, the differences were not statistically significant (Figure 5). Login to comment 150 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala We have previously shown that prazosin binding-induced conformational changes as monitored by measuring the level of binding of 5D3 to BCRP could be affected by single mutations, thereby leading to altered transport activity.15 To investigate whether the mutations of Pro392 and Pro485 cause conformational changes in BCRP that affect transport activity, we measured the level of binding of 5D3 to wild-type BCRP, P392A, or P485A in the presence and absence of varying concentrations of the substrate prazosin (0-40 μM) or MX (0-80 μM). Login to comment 153 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala In the presence of prazosin, wild-type BCRP was associated with a concentration-dependent increase in 5D3-phycoerythrin fluorescence by up to 70%; however, there was an only slight increase in 5D3-phycoerythrin fluorescence for P392A and a slight decrease in 5D3-phycoerythrin fluorescence for P485A (Figure 6A). Login to comment 155 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala The patterns of 5D3-phycoerythrin fluorescence with MX for wild-type BCRP, P392A, and P485A were similar, but different from those with prazosin (Figure 6B). Login to comment 162 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala Concentration-dependent effects of prazosin and MX on the binding of 5D3 to wild-type BCRP, P392A, and P485A. Login to comment 164 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala Shown are means ± SD of three experiments. Asterisks indicate statistically significant differences between wild-type BCRP and P392A or P485A (p < 0.05 by a Student`s t test) at various prazosin concentrations. Login to comment 180 ABCG2 p.Pro485Ala We found that alanine substitution of Pro485 had a profound impact on the substrate selectivity of BCRP. Login to comment 181 ABCG2 p.Pro485Ala P485A exhibited a significant decrease in the efflux activity of BODIPY-prazosin, but not of MX and Hoechst 33342, and conferred lower resistance to SN-38, but not to MX (Figure 4 and Table 1). Login to comment 183 ABCG2 p.Pro392Ala On the other hand, alanine substitution of Pro392 caused a significant decrease in the transport activity of all the substrates tested (Figure 4 and Table 1). Login to comment 184 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala Such changes in transport activity of P392A and P485A do not seem to be related to alterations in Figure 7. Login to comment 205 ABCG2 p.Pro485Ala Alanine substitution of Pro485 would eliminate the helical distortion of TM3 and the flexibility and influence the helical packing with neighboring TM helices, thereby affecting transport activity for some substrates. Login to comment 206 ABCG2 p.Pro485Ala The results of this study suggest that alanine substitution of Pro485 does not affect binding or transport for MX and Hoechst 33342 but weakens binding and transport for prazosin and SN-38. Login to comment 225 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala Second, the responses of wild-type BCRP, P392A, and P485A to 5D3 binding in the presence of prazosin were significantly different from each other (Figure 6A). Login to comment 226 ABCG2 p.Pro392Ala ABCG2 p.Pro485Ala However, there was no difference between wild-type BCRP, P392A, and P485A in response to 5D3 binding in the presence of MX (Figure 6B). Login to comment