ABCMdb

PMID: 16086592

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 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 47 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly All experiments detailed in this paper make use of these gain-of-function mutations, either R482T in the MCF-7/AdVp3000 cell line or R482G in the transiently transfected HeLa cells. Login to comment 49 ABCG2 p.Arg482Gly HeLa cells (cervical epitheliod carcinoma) were cultured in DMEM complete media at 37 °C. ABCG2 (R482G) was overexpressed by transient transfection using the T7 vaccinia virus system as described previously (43). Login to comment 50 ABCG2 p.Asp210Asn 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 52 ABCG2 p.Arg482Thr MCF-7 cells (breast adenocarcinoma) selected and maintained in 3 µg/ mL doxorubicin and 5 µg/mL verapamil (MCF-7/AdVp3000) overexpress ABCG2 (R482T) at the plasma membrane. Login to comment 57 ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn Cloning and Expression of ABCG2-ABCG2, ABCG2-MABCG2, ABCG2-P-ABCG2, ABCG2 (D210N), ABCG2 (D210N)-ABCG2, and ABCG2∆EC-C. Login to comment 59 ABCG2 p.Arg482Gly ABCG2-ABCG2 encodes two identical ABCG2 (R482G) proteins connected by a SacII restriction enzyme site which adds a proline-arginine linker (PR). Login to comment 60 ABCG2 p.Arg482Gly ABCG2-M-ABCG2 encodes two ABCG2 (R482G) proteins linked by the sequence (PR-NEVELENAADE- SKSEIDALEMSSNDSRSSLIRKRSTRRSVRGSQAQDR- KLSTKEALD-PR) derived from the flexible linker region of human P-glycoprotein. Login to comment 61 ABCG2 p.Arg482Gly ABCG2-P-ABCG2 encodes two ABCG2 (R482G) proteins linked by the sequence (PR- KRMKKRGVLTEKNANDPENVGERSDLSSDRKMLQ- ESSEEESDTYGEIGLSKSEAIFHWRNLCYEVQIKAE- PR) derived from the flexible linker region of yeast ABC transporter PDR5. Login to comment 64 ABCG2 p.Arg482Gly ABCG2 p.Asp210Asn ABCG2 (D210N) was constructed by site-directed mutagenesis of aspartate 210 of the parent plasmid pTM1-ABCG2 (R482G). Login to comment 65 ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 67 ABCG2 p.Arg482Gly ABCG2 p.Cys592Ala ABCG2 p.Cys603Ala ABCG2 p.Cys608Ala ABCG2∆EC-C, which contains all three endogenous extracellular cysteines replaced with alanine (C592A, C603A, C608A), was constructed by site-directed mutagenesis of the parent plasmid pTM1-ABCG2 (R482G). Login to comment 109 ABCG2 p.Arg482Gly RESULTS ABCG2 (R482G) Chimeric Dimers Are Expressed at the Cell Surface and Transport ABCG2 Substrates. Login to comment 111 ABCG2 p.Arg482Gly In all of the experiments presented here involving transient expression of ABCG2, we used the ABCG2 (R482G) variant because it has a broader substrate specificity than the wild-type protein (39-41, 46). Login to comment 112 ABCG2 p.Arg482Gly Throughout, the designation ABCG2 refers to the R482G variant unless otherwise noted. Login to comment 133 ABCG2 p.Arg482Gly 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-M-ABCG2 (black thin line), or ABCG2-P-ABCG2 (dashed line). Login to comment 136 ABCG2 p.Arg482Gly HeLa cells were co-infected/transfected in six-well plates with plasmid DNA constructs of the empty vector pTM1 (lane 1), ABCG2 (R482G) (lane 2), ABCG2-ABCG2 (lane 3), ABCG2-M-ABCG2 (lane 4), and ABCG2-P-ABCG2 (lane 5). Login to comment 153 ABCG2 p.Arg482Gly After 24 h at 32 °C, cells were harvested and incubated with the fluorescent ABCG2 (R482G) substrates rhodamine 123 or mitoxantrone for 30 min at 37 °C, followed by a second incubation for 30 min in drug-free media to allow for transporter-mediated efflux. Cellular drug accumulation was analyzed using flow cytometry. Login to comment 159 ABCG2 p.Arg482Gly A Single Mutation in the Walker B Motif of ABCG2 (R482G) Monomers and Chimeric Dimers Abrogates Function but Not Cell Surface Expression. Login to comment 160 ABCG2 p.Arg482Gly ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Arg482Gly ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 173 ABCG2 p.Arg482Thr Because the cross-linker is not cell permeable, these experiments were performed in crude membrane preparations of drug-selected MCF-7/AdVp3000 cells that overexpress a homogeneously glycosylated population of ABCG2 (R482T) (Figure 3A, lane 1). Login to comment 174 ABCG2 p.Arg482Thr Membrane preparations from these cells were used because of their uniform banding pattern on SDS-PAGE and their high level of ABCG2 (R482T) expression. Login to comment 175 ABCG2 p.Arg482Thr In the absence of the cross-linker, ABCG2 (R482T) migrates as a monomer by SDS-PAGE analysis (Figure 3A, lane 1). Login to comment 184 ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn 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 ABCG2 p.Arg482Gly ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn 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 ABCG2 p.Arg482Gly ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn 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 ABCG2 p.Asp210Asn ABCG2 p.Asp210Asn 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 195 ABCG2 p.Arg482Thr In six-well plates, MCF-7/ AdVp3000 cells overexpressing ABCG2 (R482T) were incubated with a 1 mM solution of DPDPB for 2 h at 4 °C. Login to comment 201 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr When ABCG2 (R482T) was cross-linked with DPDPB, a higher molecular mass species at a molecular mass consistent with an ABCG2 (R482T) dimer (Figure 4, lane 2) was apparent, as compared to the monomer species observed in the absence of the cross-linker (Figure 4, lane 1). Login to comment 211 ABCG2 p.Arg482Thr To determine if the extracellular cysteine residues are crucial for activity, the ABCG2∆EC-C variant was expressed in HeLa cells, and drug transport activity was measured as described in Experimental Procedures. We observed that the ABCG2∆EC-C variant had comparable levels of transport FIGURE 3: Nucleotide cross-linking of ABCG2 (R482T) in cell membranes. Login to comment 214 ABCG2 p.Arg482Thr Proteins (5 µg in panel B and 25 µg in panel C) were 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. FIGURE 4: Sulfhydryl-reactive chemical cross-linking of ABCG2 (R482T) in whole cells. Login to comment 219 ABCG2 p.Arg482Gly Crude HeLa cell membrane preparations of cells transiently overexpressing the ABCG2 and ABCG2∆EC-C were incubated in the presence (stimulated) and absence (basal) of 20 µM ABCG2 (R482G) substrate prazosin. Login to comment 220 ABCG2 p.Arg482Gly The ABCG2∆EC-C variant demonstrated drug-stimulated ATPase activity comparable to the parent protein, ABCG2 (R482G), with perhaps a slight increase in the basal activity (Figure 5D). Login to comment 235 ABCG2 p.Arg482Gly Basal (gray bars) and drug-stimulated ATPase activity (black bars) of pTM1 (negative control empty vector), ABCG2 (R482), and ABCG2∆EC-C in the ABCG2 (R482G) background. Login to comment 242 ABCG2 p.Lys86Met These data are corroborated by two studies that demonstrated co-immunoprecipitation of differentially tagged ABCG2 monomers (5, 26), including dominant-negative effects upon coexpression of wild-type ABCG2 and inactive ABCG2 molecules containing a Walker A mutation (K86M) (26) or a Leu to Pro mutation in the fifth transmembrane segment at position 554 (5). Login to comment 243 ABCG2 p.Lys86Met ABCG2 p.Asp210Asn 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 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met ABCG2 p.Asp210Asn 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