ABCMdb

ABCG2 p.Asp210Asn

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Publications

PMID: 16086592 [PubMed] Bhatia A et al: "Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers."

No. Sentence Comment

4 Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Login to comment
5 Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Login to comment
50 ABCG2 monomers, chimeric dimers, D210N variants, and extracellular cysteine-less variants were cloned into the pTM1 vector where expression was under the control of the T7 promoter. Login to comment
57 Cloning and Expression of ABCG2-ABCG2, ABCG2-MABCG2, ABCG2-P-ABCG2, ABCG2 (D210N), ABCG2 (D210N)-ABCG2, and ABCG2∆EC-C. Login to comment
64 ABCG2 (D210N) was constructed by site-directed mutagenesis of aspartate 210 of the parent plasmid pTM1-ABCG2 (R482G). Login to comment
65 ABCG2 (D210N)-ABCG2 was built using a combination of ABCG2 (D210N) and ABCG2-ABCG2; ABCG2 (D210N) was altered to include a SacII site immediately upstream of the XhoI site. Login to comment
66 The stop codon was then deleted, and the SacII to XhoI fragment from ABCG2-ABCG2 (the second gene of the chimeric dimer) was inserted in the pTM1-ABCG2 (D210N) vector. Login to comment
160 To examine the effects of mutations to the ATP binding site on ABCG2 function, we constructed a variant of the monomeric ABCG2 (R482G) in the Walker B motif (D210N), thought to be involved in magnesium binding. Login to comment
162 We evaluated cell surface expression of ABCG2 (D210N) by flow cytometry using the monoclonal antibody 5D3 that recognizes an external epitope of ABCG2 (18), followed by incubation with a FITC-conjugated secondary antibody for detection (Figure 2A), as well as by cell surface biotinylation experiments (Figure 2B). Login to comment
163 This construct, ABCG2 (D210N), is expressed at the cell surface by both of these assays but is not functional for either rhodamine 123 or mitoxantrone transport (Figure 2C,D). Login to comment
164 The ABCG2 (D210N) monomer is recognized better by the conformation-sensitive 5D3 antibody than the ABCG2 (R482G) protein, as demonstrated by the greater shift in fluorescence intensity, suggesting that the presence of the mutation may have caused a conformational change in the protein (47). Login to comment
165 We then incorporated this variant into the first gene of the chimeric ABCG2 dimer [ABCG2 (D210N)- ABCG2] and assessed cell surface expression and function by the assays described above. Login to comment
166 We determined that ABCG2 (D210N)-ABCG2 is expressed at the cell surface by both flow cytometry analysis with the 5D3 antibody (Figure 2A) and cell surface biotinylation (Figure 2B). Login to comment
167 However, the transport function of this chimera is completely abrogated (Figure 2C,D), showing a pattern similar to both the pTM1 negative control and the ABCG2 (D210N) monomer. These data indicate that two intact ATP sites are necessary for function, as has been established for numerous full-length ABC transporters, and suggest that ABCG2 functions as a dimer. Login to comment
168 We did not construct the ABCG2 chimeric dimer with the D210N mutation introduced into the second ABCG2 gene, but we predict a similar dominant-negative effect if the protein proved to fold properly. Login to comment
184 Therefore, we used the non-membrane-permeable bifunctional sulfhydryl-reactive cross-linker DPDPB [1,4-di[3'-(2'-pyridyldithio)propionamido]butane] that has a spacer length of 19.9 Å (49) to determine if these extracellular FIGURE 2: Cell surface expression and drug efflux activity of the ABCG2 (D210N) monomer and chimeric dimer ABCG2 (D210N)- ABCG2. Login to comment
186 HeLa cells were co-infected/transfected with plasmid DNA constructs of the empty vector pTM1 (gray shaded peak), ABCG2 (R482G) (black thick line), ABCG2-ABCG2 (gray thick line), ABCG2 (D210N) (black thin line), or ABCG2 (D210N)-ABCG2 (dashed line). Login to comment
189 HeLa cells were co-infected/transfected in six-well plates with plasmid DNA constructs containing ABCG2 (R482G) (lane 1), ABCG2 (D210N) (lane 2), ABCG2-ABCG2 (lane 3), and ABCG2 (D210N)-ABCG2 (lane 4). Login to comment
191 Cells were washed with buffer containing 100 mM glycine to bind excess biotin prior to cell lysis. Biotinylated proteins were solubilized in RIPA buffer and then selected with monomeric avidin beads. Cell surface proteins were denatured with 1× Laemmli sample buffer containing -ME, separated by SDS-PAGE (7.5% gel), and detected by immunoblot analysis using the monoclonal antibody BXP-21 (1:1000) as described in Experimental Procedures. (C, D) Drug accumulation in HeLa cells expressing the ABCG2 (D210N) monomer and chimeric dimer. Login to comment
193 Cells were harvested by centrifugation and incubated with drug-free media for an additional 30 min at 37 °C to allow for drug efflux and analyzed by flow cytometry. Histograms depict the cellular fluorescence intensity of the negative control pTM1 empty vector (gray shaded peak), ABCG2 (black thick line), ABCG2-ABCG2 (gray thick line), ABCG2 (D210N) (black thin line), and ABCG2 (D210N)-ABCG2 (dashed line). Login to comment
243 However, the K86M mutation appears to have a more dramatic effect on targeting of ABCG2 to the cell surface than the D210N mutation described here. Login to comment
244 By cell surface biotinylation experiments, we showed that ABCG2 (D210N) is expressed at the surface similarly to ABCG2 (R482G) (Figure 2B) whereas ABCG2 (K86M) is found predominantly in the endoplasmic reticulum with a small percentage localized to the plasma membrane (26). Login to comment

PMID: 17375082 [PubMed] Hardwick LJ et al: "The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2)."

No. Sentence Comment

42 In contrast, the introduction of a D210N replacement in the Walker B region of the first unit, which inactivated the ATPase activity of the unit, imposed a dominant-negative phenotype on the fusion proteins. Login to comment

PMID: 19691360 [PubMed] Hou YX et al: "Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport."

No. Sentence Comment

184 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
191 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

PMID: 18328253 [PubMed] Loo TW et al: "Mutational analysis of ABC proteins."

No. Sentence Comment

127 In another approach to determine if BCRP functioned as a dimer, inactive mutants were constructed by introducing a D210N change in the Walker B motif [44]. Login to comment
128 Chimeric BCRP molecules were constructed that contained two wild-type BCRP molecules fused together or a wild-type molecule fused to a D210N mutant. Login to comment
130 Expression of the chimeric molecule containing a D210N mutation, however, resulted in a dominant-negative phenotype, as the protein was expressed at the cell surface but was not functional. Login to comment