ABCMdb

ABCG2 p.Asp210Asn

Predicted by SNAP2:	A: D (85%), C: D (85%), E: D (85%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (95%), M: D (91%), N: D (85%), P: D (95%), Q: D (91%), R: D (95%), S: D (85%), T: D (85%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Oligomerization of the human ABC transporter ABCG2... Biochemistry. 2005 Aug 16;44(32):10893-904. Bhatia A, Schafer HJ, Hrycyna CA
Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers.
Biochemistry. 2005 Aug 16;44(32):10893-904., 2005-08-16 [PMID:16086592]

Abstract [show]
Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. 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. 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. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.

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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

[hide] The emerging pharmacotherapeutic significance of t... Br J Pharmacol. 2007 May;151(2):163-74. Epub 2007 Mar 20. Hardwick LJ, Velamakanni S, van Veen HW
The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2).
Br J Pharmacol. 2007 May;151(2):163-74. Epub 2007 Mar 20., [PMID:17375082]

Abstract [show]
The breast cancer resistance protein (also termed ABCG2) is an ATP-binding cassette transporter, which mediates the extrusion of toxic compounds from the cell, and which was originally identified in relation to the development of multidrug resistance of cancer cells. ABCG2 interacts with a range of substrates including clinical drugs but also substances such as sterols, porphyrins and a variety of dietary compounds. Physiological functions of ABCG2 at both cellular and systemic levels are reviewed. For example, ABCG2 expression in erythrocytes may function in porphyrin homeostasis. In addition, ABCG2 expression at apical membranes of cells such as hepatocytes, enterocytes, endothelial and syncytiotrophoblast cells may correlate to protective barrier or secretory functions against environmental or clinically administered substances. ABCG2 also appears influential in the inter-patient variation and generally poor oral bioavailability of certain chemotherapeutic drugs such as topotecan. As this often precludes an oral drug administration strategy, genotypic and environmental factors altering ABCG2 expression and activity are considered. Finally, clinical modulation of ABCG2 activity is discussed. Some of the more recent strategies include co-administered modulating agents, hammerhead ribozymes or antisense oligonucleotides, and with specificity in cell targeting, these may be used to reduce drug resistance and increase drug bioavailability to improve the profile of chemotherapeutic efficacy versus toxicity. While many such strategies remain in relative infancy at present, increased knowledge of modulators of ABCG2 could hold the key to novel approaches in medical treatment.

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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

[hide] Effects of putative catalytic base mutation E211Q ... Biochemistry. 2009 Sep 29;48(38):9122-31. 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 [PMID:19691360]

Abstract [show]
ABCG2 is a half-ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a transmembrane domain. This half-ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. 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. However, whether one ATP hydrolysis or two ATP hydrolyses in the homodimer of ABCG2 with the NBD.ATP.ATP.NBD sandwich structure is/are required for the ATP-dependent solute transport is not known yet. 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. Although membrane vesicles prepared from BHK cells expressing YFP/ABCG2 exert higher ATPase activity than that of wt ABCG2, the dATP-dependent methotrexate transport activities of these two proteins are the same. 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. Furthermore, the membrane vesicles containing both YFP/ABCG2 and E211Q exert approximately 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.

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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

[hide] Mutational analysis of ABC proteins. Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5. Loo TW, Clarke DM
Mutational analysis of ABC proteins.
Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5., [PMID:18328253]

Abstract [show]
The 49 human members of the ATP-binding cassette (ABC) family of proteins are involved in a wide range of activities such as active transport of compounds across membranes, extraction of compounds out of membranes, functioning as ion channels, or regulators of channel activity. Mutations and/or overexpression of many of the proteins can have adverse effects on health. A goal in the study of ABC proteins is to understand their mechanisms of action. This review will focus on the mutational approaches that have been used to study the structure and mechanisms of some ABC proteins.

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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