ABCMdb

PMID: 16618113

Polgar O, Ozvegy-Laczka C, Robey RW, Morisaki K, Okada M, Tamaki A, Koblos G, Elkind NB, Ward Y, Dean M, Sarkadi B, Bates SE
Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization.
Biochemistry. 2006 Apr 25;45(16):5251-60., 2006-04-25 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. Login to comment 7 ABCG2 p.Gly553Glu Similar results were observed with the G553E mutant. Login to comment 8 ABCG2 p.Gly553Leu Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein. Login to comment 9 ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each other but are unable to complete normal trafficking. Login to comment 10 ABCG2 p.Gly553Leu Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Login to comment 48 ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu The G553L and G553E mutants were generated by site-directed mutagenesis in the pcDNA3.1/ Myc-HisA(-) vector (Invitrogen) as previously described (23). Login to comment 82 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu N-Terminally GFP-tagged wild-type (wt) ABCG2 and ABCG2-G553L constructs were generated by cloning the XhoI-BamHI fragment of pAcUW21-L/wtABCG2 or pAcUW21-L/ABCG2-G553L into the corresponding site of the pEGFP vector (Clontech, Mountain View, CA). Login to comment 83 ABCG2 p.Gly553Leu HEK 293 cells were transfected with the pEGFP-wtG2 or pEGFP-G553L vector using the FuGene reagent (Roche, Indianapolis, IN). Login to comment 86 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu Generation of Sf9 Cells Expressing the Wild Type or the K86M or G553L Mutant. Login to comment 87 ABCG2 p.Lys86Met Generation of transfer vectors containing wt ABCG2 or K86M has been described previously (26, 27). Login to comment 88 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu The transfer vector carrying the G553L mutant was generated by cloning the SacI fragment of pcDNA 3.1/G553L into the corresponding site of the pAcUW21-L vector. Login to comment 101 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu To study the function of wt ABCG2 when coexpressed with the G553L mutant, 4 × 106 Sf9 cells were transfected with the combination of different volumes of recombinant baculoviruses (as indicated in Figure 9) containing wt ABCG2, G553L, K86M, or -galactosidase. Login to comment 108 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu Sf9 membranes containing wild-type ABCG2, G553L, or K86M were harvested, and membranes were isolated and stored at -80 °C according to the method of Sarkadi et al. (29). Login to comment 114 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu To begin to characterize the residue, HEK 293 cells were stably transfected with ABCG2 carrying a glycine to leucine substitution at amino acid 553 (G553L) using a pcDNA3 vector. Login to comment 118 ABCG2 p.Gly553Leu unlike the wild-type protein, revealed no surface expression for the G553L mutant (Figure 2A, six of the 24 clones shown). Login to comment 122 ABCG2 p.Gly553Leu As illustrated in panels B and C of Figure 2, the G553L mutant was represented by a double band on immunoblot with the majority of the protein running lower than the normal molecular mass of 72 kDa. To investigate whether this lower-molecular mass band was representative of the nonglycosylated protein, isolated membranes from cells bearing wild-type or mutant vectors were treated with the PNGase F enzyme to remove N-linked glycans. Login to comment 123 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu In the case of the G553L mutant, no significant shift in molecular mass was observed after PNGase F treatment (Figure 3), indicating that, indeed, the G553L mutant is underglycosylated, while a clear shift to a lower-molecular mass band was seen in membranes extracted from HEK 293 cells transfected with wild-type ABCG2 or from flavopiridol-selected MCF-7 cells used as controls (25). Login to comment 124 ABCG2 p.Gly553Leu Since the G553L mutant did not demonstrate normal surface localization by flow cytometry with the 5D3 antibody, immunofluorescence studies were carried out with the BXP-21 antibody on the clone with the highest expression level (clone 2 in Figure 2B). Login to comment 125 ABCG2 p.Gly553Leu Figure 4 demonstrates that wild-type ABCG2 is localized to the cell membrane, while the FIGURE 2: Surface expression and protein and RNA levels of the G553L mutant transfected to HEK 293 cells. Login to comment 126 ABCG2 p.Gly553Leu (A) Flow cytometry with the 5D3 surface antibody for six of the G553L clones. Login to comment 128 ABCG2 p.Gly553Leu The G553L mutant is not detectable on the cell surface, while the wild-type protein is localized to the surface. Login to comment 129 ABCG2 p.Gly553Leu (B) Membrane proteins from the same G553L clones (25 µg/lane) were separated by SDS-PAGE, transferred onto a PVDF membrane, and probed with the monoclonal anti-ABCG2 antibody BXP-21. Login to comment 135 ABCG2 p.Gly553Leu One hundred micrograms of membranes was incubated with 3 µL of PNGase F overnight at 37 °C followed by SDS-PAGE separation and immunoblotting with the BXP-21 monoclonal anti-ABCG2 antibody, resulting in a clear shift to a lower-molecular mass band in the wild-type ABCG2-transfected and the flavopiridol-selected MCF-7 control cell lines (MCF-7/FLV1000), while there is no significant shift in the case of the G553L mutant. Login to comment 136 ABCG2 p.Gly553Leu G553L mutant is predominantly intracellular. Login to comment 137 ABCG2 p.Gly553Leu Parallel staining with the anti-calnexin monoclonal antibody revealed that the G553L mutant colocalizes with the endoplasmic reticulum (ER) chaperone, calnexin. Login to comment 138 ABCG2 p.Gly553Leu These results suggest that the G553L mutant is not able to complete normal folding or processing to move to the cell surface in mammalian cells. Login to comment 141 ABCG2 p.Gly553Leu Unexpectedly, using the homobifunctional amine-reactive cross-linkers MBS (9.9 Å arm length) and DSG (7.7 Å arm length) on intact cells, the G553L mutant could be cross-linked as indicated by the appearance of higher-molecular mass bands corresponding to dimers and higher-order multimers of the 72 kDa ABCG2 monomer (Figure 5). Login to comment 145 ABCG2 p.Gly553Leu As the lower protein expression levels seen upon immunoblotting and the lack of surface expression might be the result of abnormal folding and/or dimerization, using the baculovirus heterologous expression system, we expressed the G553L mutant in Sf9 insect cells (S. frugiperda ovarian cells), which might be more tolerant of an aberrant protein and typically yields higher levels of protein expression (34). Login to comment 146 ABCG2 p.Gly553Leu Interestingly, we found that the G553L mutant migrates to the cell surface in the insect cells as demonstrated by flow cytometry with the 5D3 antibody (Figure 6A). Login to comment 148 ABCG2 p.Lys86Met The ATPase activity was comparable to that of the nonfunctional K86M mutant (26) (Figure 6B). Login to comment 149 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu FIGURE 4: Localization of the G553L mutant in HEK 293 cells. Confocal microscopy of stably transfected HEK 293 reveals that the G553L mutant colocalizes with the ER marker calnexin, while the wild-type protein localizes to the cell surface. Login to comment 154 ABCG2 p.Gly553Leu Cross-linking is observed with both amine-reactive cross-linkers in HEK 293 cells transfected with either wild-type ABCG2 or the G553L mutant as suggested by dimers and higher-order multimers. Login to comment 155 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu The molecular mass of monomeric wild-type ABCG2 is 72 kDa. To further investigate whether the G553L mutation interferes with dimerization, we transiently expressed a GFP-tagged G553L mutant together with the wild-type protein in HEK 293 cells and performed confocal microscopy. Login to comment 157 ABCG2 p.Gly553Leu As shown in Figure 7, the GFP-tagged G553L mutant is localized to the ER as before and the nontagged wild-type protein trafficked to the surface, suggesting that the mutant was unable to form competent dimers with the wild-type protein. Login to comment 159 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu Figure 8 represents Hoechst 33342 transport activities for Sf9 cells coinfected with a constant amount of recombinant baculovirus carrying wild-type ABCG2 together with varying amounts of the G553L and K86M mutants. Login to comment 160 ABCG2 p.Gly553Leu In case of the 50:400 ratio, which represents approximately equal protein expression levels upon immunoblotting for the wild-type and the G553L mutant (data not shown), an ~35% decrease in transport activity is seen and suggests some interaction between the wild-type and mutant proteins. Login to comment 161 ABCG2 p.Lys86Met However, the impact is much smaller than that observed with K86M, a mutation in Walker A that has been reported to retain dimerization with a functional dominant negative effect (35). Login to comment 162 ABCG2 p.Gly553Glu Evaluating the effect of a charged residue at position 553, we replaced the glycine with glutamic acid (G553E) and transfected HEK 293 cells. Login to comment 164 ABCG2 p.Gly553Glu Figure 9 shows that, like the leucine mutant, the G553E mutant did not move to the cell surface as verified by no staining with the 5D3 surface antibody on flow cytometry (Figure 9A). Login to comment 166 ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu The G553E mutant, like the G553L mutant, is represented by a double band on immunoblots, suggestive of impaired glycosylation, and in fact, following treatment with PNGase F, only one lower-molecular mass band was visible upon immunoblotting (data not shown). Login to comment 169 ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu We found that substitution of glycine 553 with either leucine (G553L) or glutamic acid (G553E) followed by transfection into HEK 293 cells resulted in a markedly reduced level of protein expression with impaired glycosylation and predominant ER retention. Login to comment 170 ABCG2 p.Gly553Leu In SF9 insect cells, the G553L mutant trafficked to the cell surface but, nevertheless, was unable to hydrolyze ATP. Login to comment 171 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu Although the wild-type protein was unable to rescue the mutant from its ER localization in the mammalian cells, chemical cross-linking studies indicated that the two G553L monomers are FIGURE 6: G553L mutant in Sf9 insect cells. Login to comment 172 ABCG2 p.Gly553Leu (A) Wild-type ABCG2 and the G553L mutant are detectable on the surface of Sf9 cells with the 5D3 monoclonal anti-ABCG2 antibody on flow cytometry (as detailed in Figure 2). Login to comment 173 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu (B) The G553L mutant displays basal ATPase activity similar to that of the nonfunctional K86M mutant in Sf9 membranes. Login to comment 177 ABCG2 p.Gly553Leu When Sf9 insect cells were coinfected with the G553L mutant and the wild-type protein, the Hoechst transport activity was reduced by approximately 35%. Login to comment 190 ABCG2 p.Gly553Leu The fact that the ABCG2 G553L mutant protein does not leave the ER in mammalian cells suggests that it is recognized by the ER`s quality control system and is degraded, explaining the low observed expression levels (38). Login to comment 192 ABCG2 p.Gly553Leu ABCG2 p.Gly553Glu In addition to the above-mentioned aberrant localization in the mammalian cells, the glycosylation pattern of the G553L and G553E mutants was also altered. Login to comment 193 ABCG2 p.Gly553Leu ABCG2 p.Gly553Leu Interestingly, FIGURE 7: Coexpression of wild-type ABCG2 and the G553L mutant in HEK 293 cells. Confocal microscopy results of HEK 293 cells transiently transfected with either the GFP-tagged G553L mutant (first row) or the GFP-tagged wild type (third row) or cotransfected with the GFP-tagged mutant and nontagged wild type (second row) are presented. Login to comment 195 ABCG2 p.Gly553Leu The cotransfection images (second row) show that despite the presence of the wild-type protein the G553L mutant is retained intracellularly, while the wild-type protein localizes to the cell surface. Login to comment 198 ABCG2 p.Gly553Leu In the case of ABCG2, previous studies have shown that the protein is glycosylated at asparagine 596 and that the glycosylation is not necessary for cell surface localization (39) or function, suggesting that the G553L mutant is not retained in the ER merely due to failure to undergo glycosylation. Login to comment 199 ABCG2 p.Gly553Leu The fact that we found no evidence of colocalization on confocal microscopy when the G553L mutant was coexpressed with wild-type ABCG2 in HEK 293 cells suggests that there is no stable interaction between wild-type and mutant proteins in the ER of mammalian cells, as would be expected if the wild-type and mutant formed stable dimers. Login to comment 203 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu Sf9 cells were infected with a combination of the indicated volumes of recombinant baculoviruses containing wild-type ABCG2, G553L, K86M, or -galactosidase. Login to comment 207 ABCG2 p.Lys86Met Each column represents the average of three measurements (except for the 50:200 wt + K86M column, where only one measurement was performed). Login to comment 209 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu The G553L mutant shows no transport of Hoechst 33342 (second column) and at the 50:400 ratio (fifth column), representing approximately equal protein expression levels for the mutant and the wild type, results in a 35% decrease in activity, while in case of the K86M mutant, the same ratio (last column) almost completely abrogates Hoechst transport. Login to comment 210 ABCG2 p.Gly553Glu FIGURE 9: Surface expression of the G553E mutant in HEK 293 cells and cross-linking with DSG. Login to comment 211 ABCG2 p.Gly553Glu (A) The G553E mutant (six clones are shown) is not detectable on the cell surface with the 5D3 antibody by flow cytometry performed as described in the legend of Figure 2: negative control antibody (s) or 5D3 antibody (- - -). Login to comment 212 ABCG2 p.Gly553Glu (B) Cross-linking (as detailed in Figure 5) is observed following treatment of transfected HEK 293 cells with DSG in case of both the wild type (50 µg of protein) and the G553E mutant (100 µg of protein). Login to comment 216 ABCG2 p.Gly553Leu Though the G553L mutant is confined to the ER, it must be near another mutant protein because chemical cross-linking of two mutant proteins even at a distance of 7.7 Å was observed. Login to comment 222 ABCG2 p.Lys86Met Although results with the catalytically inactive control K86M mutant were not precisely as expected on the basis of the ratios transfected, the marked reduction in Hoechst transport activity is consistent with the reported dominant negative effect for this Walker A mutant (35). Login to comment 223 ABCG2 p.Lys86Met ABCG2 p.Gly553Leu In contrast to the results observed with the K86M mutant-wild-type dimer, a roughly 35% decrease was observed in the Sf9 cells expressing both the wild-type and the G553L mutant protein. Login to comment 224 ABCG2 p.Gly553Leu This limited, but reproducible, effect of the G553L mutant on the wild-type protein argues that the mutant has a conformation that can associate with the wild-type protein, although that association must be relatively weak. Login to comment 228 ABCG2 p.Gly553Glu The G589E mutation in the white protein, analogous to our ABCG2 G553E mutant, apparently disrupts the white-brown heterodimer as suggested by the significantly reduced red eye pigment levels. Login to comment 229 ABCG8 p.Gly574Glu Furthermore, the naturally occurring ABCG8 G574E mutant, carrying a mutation corresponding to phenylalanine 551 of ABCG2, was also characterized as interfering with dimerization. Login to comment 230 ABCG8 p.Gly574Glu When the ABCG8 G574E mutant was coexpressed with wild-type ABCG5 in CHO-K1 cells, no protein in the position of a dimer was detected on native gels (37). Login to comment 231 ABCG8 p.Gly574Arg On the other hand, the ABCG8 G574R mutant, although having some effect on ABCG8 maturation, did not prevent heterodimerization (33). Login to comment 233 ABCG2 p.Leu554Pro However, in the case of ABCG2, the inactive L554P mutant was reported to be able to partially reverse the drug resistance of PA317 cells cotransfected with the mutant and wild-type proteins, a result implying that residue 554 is critical for function, yet mutating this residue does not prevent dimerization. Login to comment