ABCMdb

ABCG2 p.Lys86Met

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PMID: 12374800 [PubMed] Ozvegy C et al: "Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation."

No. Sentence Comment

2 In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Login to comment
4 We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. Login to comment
44 As a control, we mutated a crucial amino acid in the catalytic center of ABCG2-R482G (Lys-86 was changed to Met) in hope of producing a non-functional transporter. Login to comment
60 Wild-type ABCG2 (Arg-482) and its variants (R482T and K86M) were created using ABCG2-R482G cDNA as a template (4) by overlap extension PCR (30). Login to comment
61 Two internal complementary primer pairs were used with each containing the specific mutation as follows: the Arg- primer pairs were 5Ј-TTATTACCAATGCGCATGTTACC-3Ј and 5Ј-GG- TAACATGCGCATTGGTAATAA-3Ј; the R482T primer pairs were 5Ј-T- TATTACCTATGACGATGTTACC-3Ј and 5Ј-GGTAACATCGTCATAG- GTAATAA-3Ј; and the K86M primer pairs were 5Ј-TGGAGGCATGTC- TTCGTTATT-3Ј and 5Ј-TAATAACGAAGACATGCCTCCA-3Ј, respectively. Login to comment
62 The two outer primer pairs were 5Ј-CTTGGGATACTTGAATC- AGC-3Ј and 5Ј-GGTCATGAGAAGTGTTGCTA-3Ј for the wild-type and the amino acid 482 variants and 5Ј-GTATATTAATTAAAATACTATA- CTG-3Ј and 5Ј-GGCTCATCCAAGAACAAGAT-3Ј for the K86M mutant. Login to comment
64 The PCR products containing the Arg-482 or R482T coding sequence were digested with PstI and MscI enzymes and ligated between the corresponding sites of the pAcUW21-L/ABCG2 vector. The PCR product coding for the K86M variant was digested with NotI and SpeI enzymes and ligated to the NotI and SpeI sites of the pAcUW21-L/ABCG2 (R482G) vector. The mutations were confirmed by sequencing the PstI-MscI or the NotI-SpeI fragments of the constructs, respectively. Login to comment
108 In the present study we utilized this expression system to produce the wild-type human ABCG2 protein and its variants, R482G and R482T, as well as a catalytic center mutant, K86M. Login to comment
109 Site-directed mutagenesis was performed on a human ABCG2 cDNA, which possesses Gly at position 482 (4), to create the wild-type (Arg-482) and the R482T and K86M variants of the ABCG2 half-transporter. Login to comment
125 We found equal expression levels for the wild-type, R482G, R482T, and K86M/ R482G mutant variants (data not shown). Login to comment
132 Lane 1, R482T/Sf9; lane 2, wild-type ABCG2/Sf9; lane 3, R482G/Sf9; lane 4, K86M-R482G/Sf9; lane 5, wild-type ABCG2/ HL60; lane 6, wild-type ABCG2/HL60 grown in the presence of 2.5 ␮g/ml tunicamycin; and lane 7, beta-galactosidase/Sf9. Login to comment
138 In Sf9 cells, expressing the K86M mutant, MX uptake is significantly higher and reaches the level of MX accumulation found in the FTC-inhibited cells. Login to comment
140 These experiments indicate that wtABCG2 and the R482G or R482T variants actively extrude MX in the intact Sf9 cells, whereas the K86M mutant is inactive in this respect. Login to comment
141 Prazosin, a known substrate of ABCG2 (16, 37), also increased the level of MX accumulation in insect cells expressing the active ABCG2 proteins, whereas it had no effect on the cells expressing the K86M mutant (see Fig. 2A). Login to comment
147 As shown in Fig. 3, we found that insect cells expressing the wild-type ABCG2 or the K86M mutant accumulated significantly more rhodamine 123 than cells expressing the R482G or R482T variants. Login to comment
158 Fig. 4A shows a typical Hst dye uptake experiment using intact Sf9 cells harvested post-40 h of recombinant baculovirus infection, expressing either ABCG2-R482G or its K86M mutant variant. Login to comment
161 As documented, Hst dye uptake is significantly slower in the Sf9 cells that express the ABCG2-R482G protein (Fig. 4A, line A) than in cells expressing the K86M/R482G mutant (line B). Login to comment
164 However, there is no change in the rate of Hst accumulation in cells expressing the K86M/R482G mutant (Fig. 4A, line B) or beta-galactosidase (not shown). Login to comment
168 The K86M mutant was found to be inactive at all Hst dye concentrations examined (Fig. 4C). Login to comment
173 In the present study we have compared the ATPase activity of the wild-type human ABCG2 and its variants (R482G, R482T, and K86M) in the presence and absence of a variety of potential ABCG2 substrates or inhibitors. Login to comment
174 As shown in Fig. 5A, the basal, vanadate-sensitive ATPase activity was significantly higher in membranes containing any of the wtABCG2 or its amino acid 482 variants than in those containing ABCG2-K86M/R482G or beta-galactosidase (not shown here). Login to comment
175 Despite the similar ABCG2 expression levels, this basal ATPase activity was ϳ1.5-fold higher in case of the R482G variant (71 Ϯ 10.8 nmol of Pi/mg of membrane protein/ min) than in case of the wild-type ABCG2 or the R482T variant (45 Ϯ 8.85 and 46 Ϯ 9.04 nmol of Pi/mg of membrane protein/ min, respectively) and negligible in the ABCG2-K86M mutant (5 Ϯ 0.5 nmol of Pi/mg of membrane protein/min). Login to comment
182 It is worthwhile to note that Hst had no effect on the ATPase activity of Sf9 membranes containing ABCG2-K86M or beta-galactosidase (data not shown). Login to comment
192 A, MX accumulation in Sf9 cells expressing beta-galactosidase (beta-gal), wild-type ABCG2 (Arg-482), and the R482G, R482T, or K86M mutants. Login to comment
214 We found that 8-azido-ATP binding was similar in the wild-type and in the R482G, R482T, and K86M mutant variants under these conditions (Fig. 6B). Login to comment
221 As documented in Fig. 7B, the functionally inactive ABCG2-K86M/R482G did not show any nucleotide trapping activity under these conditions (lane 5). Login to comment
224 Rhodamine 123 accumulation in Sf9 cells expressing the wild-type (Arg-482), R482G, R482T, or K86M/R482G variants of ABCG2. Login to comment
237 DISCUSSION In this paper we describe the expression and detailed functional analysis of the wild-type human ABCG2 multidrug transporter and its mutant variants R482G, R482T, and K86M. Login to comment
247 As documented under "Results," we obtained a uniformly high level expression of the wtABCG2 and its amino acid 482 variants, and as a negative control also expressed a Walker A Lys mutant of this protein (K86M). Login to comment
248 We introduced the K86M mutation into the R482G variant of ABCG2, because we expected that the mutation of the key Lys will abolish the function of ABCG2 regardless the amino acid found in position 482. Login to comment
254 A shows the increase in fluorescence due to the uptake of 2.5 ␮M Hst in ABCG2-R482G- (line A) or ABCG2-K86M/R482G (line B) -expressing Sf9 cells. Login to comment
255 B and C, the rate of Hst influx (⌬ fluorescence/⌬ time) into Sf9 cells expressing the wtABCG2 or the R482T mutant (B) and R482G or K86M/R482G (C) was determined at different Hst concentrations with or without the ABCG2 inhibitor FTC. Login to comment
258 ATPase activity measured in membranes of Sf9 cells expressing the wild-type, R482G, R482T, or K86M/R482G variants of human ABCG2. Login to comment
267 The K86M mutant of ABCG2/R482 was already investigated for Hoechst 33342 transport in mammalian cells (35), and it was found to be inactive. Login to comment
283 [␣-32 P]8-azido-ATP binding of the wild-type and R482G, R482T, or K86M/R482G mutant ABCG2 proteins. Login to comment
290 [␣-32 P]8-azido-ATP trapping of the R482G and K86M/ R482G mutant ABCG2 proteins. Login to comment
291 Sf9 membranes containing ABCG2R482G (see A, lane 2, and B, lanes 1-3), ABCG2-K86M (B, lane 5), or beta-galactosidase (beta-gal) (see A, lane 1 and B, lane 4) were incubated for 5 min at 37 °C with 5 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate (except for lane 1) and 2 mM Mg2ϩ (A) or 2 mM Co2ϩ (B) as described under "Experimental Procedures." Login to comment
321 We have documented that the 8-azido-ATP labeling of the wtABCG2 and its amino acid 482 variants and that of the K86M mutant was similar, which is to say that they seem to have similar ATP binding capacities. Login to comment
323 Clearly, the K86M mutant was unable to form the transition state intermediate, in agreement with the inactivity of this mutant in the ATPase and transport measurements. Login to comment

PMID: 14576842 [PubMed] Doyle LA et al: "Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)."

No. Sentence Comment

87 These investigators demonstrated that a mutation of BCRP (K86M) in the Walker A nucleotide-binding domain lacked transporter activity, and served as an effective dominant-negative inhibitor of BCRP function when cotransfected with nonmutated BCRP (Ozvegy et al., 2002). Login to comment

PMID: 14645676 [PubMed] Nakanishi T et al: "Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis."

No. Sentence Comment

242 The latter is in agreement with previous observations of dominant-negative BCRP constructs possessing a mutation in the Walker A motif (K86M) (Ozvegy et al., 2002) or putative 5th transmembrane domain (L553P) (Kage et al., 2002), supporting the notion that the active form of BCRP is a homodimer. Login to comment

PMID: 14750175 [PubMed] Mizuarai S et al: "Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2."

No. Sentence Comment

212 Several mutagenesis studies on ABC transporters have shown that introduced mutations in the ABC domain completely disrupted ATPase activity.30,38,39 For example, an induced mutation in ABCG2 prevented K86M from transporting ABCG2 substrates due to the loss of ATPase activity.30 When measured in the Sf9 membrane containing ABCG2 transporters, ATPase activity of Q141K was 1.3-fold lower than that of wild-type ABCG2, although the effect on ATPase activity was relatively moderate compared to that of the introduced mutation at K86M. Login to comment
213 While K86M was located at the walker catalytic region in the ABC domain, Q141K was located between the walkerA and walkerB regions. Login to comment

PMID: 15557326 [PubMed] Ozvegy-Laczka C et al: "Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface."

No. Sentence Comment

5 The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. Login to comment
8 In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Login to comment
53 Sf9 cells expressing the ABCG2 protein or its K86M variant were prepared as described previously (31). Login to comment
54 In the present study we used the K86M variant introduced into the wild-type (R482) ABCG2 by cloning the NotI-SpeI fragment of pAcUW21-L/K86M-R482G (31) into the corresponding site of the pAcUW21-L/R482 vector. Login to comment
83 ATPase Activity Measurement-Sf9 membranes containing human ABCG2, MDR1, or ABCG2-K86M were harvested, and their membranes were isolated and stored at -80 °C according to Sarkadi et al. (34, 35). Login to comment
92 Panel A shows expression of human, wild-type ABCG2, or the K86M-ABCG2 variant in isolated membranes of Sf9 insect cells (11). Login to comment
93 Panel B shows BXP-21 immunoreactions with cell lysates of PLB cells, engineered to express the wild-type ABCG2 or its K86M mutant variant. Login to comment
94 The expression level of the K86M variant of ABCG2 was about one-third the expression obtained for the wild-type protein (these cells could not be selected by mitoxantrone; see "Experimental Procedures" and Ozvegy et al. (31)). Login to comment
110 Sf9 membranes containing WT ABCG2, ABCG2-K86M, or beta-galactosidase (beta-gal, panel A) and cell lysates from PLB (panel B), HEK 293 (panel C), and MCF-7/MX (panel D) cells expressing WT ABCG2 (or ABCG2-K86M) or parental cells (ctr.) Login to comment
131 There was no effect of 5D3 antibody on the ATPase activity of MDR1 or ABCG2-K86M membranes. Login to comment
146 Sf9 membranes containing WT ABCG2, ABCG2-K86M, or MDR1 were incubated with 20 (low 5D3, hatched columns) or 160 ␮g (high 5D3, black columns)/mg membrane concentration of 5D3 antibody. Login to comment
182 Effects of Substrates, Inhibitors, and ATP Depletion on 5D3 Reactivity in the Mutant, Non-functional K86M-ABCG2, Expressed in Intact Cells-In the next set of experiments we studied intact mammalian cells expressing a non-functional mutant (K86M) variant of ABCG2. Login to comment
184 As shown in Fig. 6, panels A and B, this K86M-ABCG2 had no MX extrusion function but showed a well measurable 5D3 binding on the cell surface. Login to comment
185 In these studies we found that the 5D3 binding of the K86M mutant ABCG2 was significantly increased by PFA fixation, ATP depletion, or Ko143 treatment. Login to comment
187 Thus, the non-functional K86M variant of ABCG2 showed a relatively high 5D3 binding in its native state, but in the case of ATP removal and Ko143 treatment, similar conformational changes were detected by 5D3 in this mutant variant as in the wild-type protein. Login to comment
188 The lack of the formation of a transition-state intermediate in the K86M-ABCG2 correlated with the absence of an effect of sodium orthovanadate. Login to comment
189 Effects of Nucleotides and Transport Inhibitors on 5D3 Reactivity of ABCG2 in Isolated Membrane Fragments-In the following experiments we examined the effects of various nucleotides and transport inhibitors on 5D3 binding by human ABCG2 and its mutant (K86M) variant in isolated insect cell membrane fragments. Login to comment
194 Fig. 7, B and C, documents the effects of various ligands on 5D3 binding to wild-type (panel B) or K86M (panel C) ABCG2 in isolated membranes. Login to comment
209 Fig. 7, panel C, shows 5D3 binding in isolated membranes containing the K86M, non-functional mutant ABCG2 protein. Login to comment
213 These data can be interpreted to mean that although MgAMP does not show binding to the protein, MgATP, MgADP, and MgAMP-PNP are bound to K86M-ABCG2, and in the absence of a full catalytic cycle, they fix the transporter in a nucleotide-bound, reduced 5D3 binding state. This fixation does not require the presence of vanadate. Login to comment
215 Interestingly, Ko143 can still stabilize the K86M-ABCG2 variant in a high 5D3 binding state. Login to comment
220 These effects were similar both in the wild-type ABCG2 and the K86M mutant variant (not documented in detail). Login to comment
237 In the present experiments the arrest of the ABCG2 transport cycle by Ko143 by the removal of the energy donor substrate, ATP, as well as by sodium orthovanadate was documented by the lack of active MX extrusion in the FIG. 6. Flow cytometry detection of the K86M-ABCG2 protein. Login to comment
238 5D3 mAb binding (panel A) and MX extrusion (panel B) in K86M mutant ABCG2-expressing intact PLB cells is shown. Login to comment
250 When examining the binding of the 5D3 antibody in intact cells to a non-functional ABCG2 catalytic center mutant (K86M-ABCG2), we found that 5D3 binding to this mutant protein was also efficient. Login to comment
259 In the K86M-ABCG2 variant the addition of MgATP, MgADP, and MgAMP-PNP all caused a major reduction of 5D3 binding, which was not further modulated by sodium orthovanadate. Login to comment
263 Interestingly, in Sf9 membranes containing either wild-type ABCG2 or its K86M mutant in the absence of Mg2ϩ (that is, in the presence of EDTA), ATP, ADP, and AMP-PNP caused a decrease in 5D3 binding (not shown in detail). Login to comment
270 Isolated membrane fragments (45 ␮g) from Sf9 cells containing WT ABCG2, ABCG2-K86M, or MDR1 were labeled with 1 ␮g/ml 5D3 (black columns) or 1 ␮g/ml mouse IgG2b as isotype control (white columns). Login to comment
273 Panel C, 5D3 binding to membranes containing K86M mutant ABCG2. Login to comment
274 Sf9 membranes containing wild-type ABCG2 (panel B) or K86M mutant ABCG2 (panels C) were incubated with 5D3 antibody in the presence of 10 mM MgAMP, MgADP, MgATP, MgAMP-PNP, MgAMP plus 2 mM vanadate, MgATP plus 2 mM vanadate, MgATP plus 2 mM vanadate plus 1 ␮M Ko143, MgADP plus 1 ␮M Ko143, MgATP plus 1 ␮M Ko143, or 10 mM MgAMP-PNP plus 1 ␮M Ko143. Login to comment

PMID: 15670731 [PubMed] Ozvegy-Laczka C et al: "Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition."

No. Sentence Comment

44 2.2. Generation of transfer vectors possessing different human ABCG2 cDNAs pAcUW21-L/ABCG2 (wild-type, R482G, T or K86M/ R482G) was constructed as described earlier [25]. Login to comment
45 In this study, we used the K86M-R482 single mutant, which was generated by cloning the NotI-SpeI fragment of pAcUW21-L/K86M-R482G [25] into the corresponding site of pAcUW21-L/R482. Login to comment
65 2.6. Measurement of [3 H]methotrexate transport by ABCG2 Sf9 membrane vesicles (90 Ag) containing one of the Arg482 mutants, wtABCG2 or the K86M mutant were prepared on the same day to ensure the same inside-out vesicle ratio of the different membranes. Login to comment
85 Similar membrane localization was found for the inactive K86M human ABCG2 mutant [25] in the insect cells. Login to comment
91 Fig. 2 demonstrates that all seven, new ABCG2-R482 mutants showed a significantly higher, vanadate-sensitive, basal ATPase activity than the K86M mutant. Login to comment
96 Vanadate-sensitive ATPase activity measured in membranes of Sf9 cells, expressing the wild-type ABCG2 or its R482 or K86M mutants. Login to comment
123 Panel B: Concentration dependence of MTX uptake by wtABCG2, R482I, R482K and K86M. Login to comment
124 Sf9 membrane vesicles (90 Ag) containing wtABCG2 (solid square), K86M (open square), R482I (diamond), and R482K (cross) were incubated in the presence or absence of 4 mM MgATP, with (up-triangle) or without 1 AM Ko143, with different MTX concentrations (10-3000 AM in a final volume of 150 Al) at 37 8C for 5 min. Login to comment
134 On the other hand, MTX accumulation measured in membranes from cells expressing h-galactosidase (Fig. 3A) or ABCG2-K86M (see below) was very low, and did not increase during this time period. Login to comment
136 MTX transport by ABCG2 was found to be fully inhibited by Ko143, down to the level of that seen in the presence of the ABCG2-K86M mutant (see Fig. 3B). Login to comment
138 Mitoxantrone and rhodamine 123 accumulation in Sf9 cells expressing wild-type ABCG2 (Arg-482), the R482X or K86M mutants. Login to comment
145 We found that none of the Arg-482 mutants had any measurable MTX uptake, as compared to the inactive ABCG2-K86M mutant (see Fig. 3B for R482I and for R482K; the data for the other variants are not shown). Login to comment
151 In order to characterize the transport of MX or R123 by the Arg-482 mutants, we have used intact insect cells expressing one of the nine 482 mutants, the wtABCG2, or the K86M mutant (as a negative control). Login to comment
155 We found that cells containing wtABCG2 or R482G, I, M, S, T, D, N, and Y mutants accumulate less mitoxantrone than cells expressing the inactive K86M mutant. Login to comment
156 However, in each case, when ABCG2 function was blocked by Ko143, MX accumulation increased to the level observed in the ABCG2-K86M expressing cells. Login to comment
159 We found that while the R482K, R482Y, the wtABCG2, and the inactive K86M mutant had no R123 extrusion activity, several ABCG2 variants were highly active in R123 extrusion. Login to comment
160 R123 transport by all these active forms was inhibited by Ko143, down to the level of the K86M mutant. Login to comment
183 All Arg-482 variants (including R482G, T, wild-type, and K86M, as a negative control) were expressed in insect cells, which produce high levels of ABCG2 and allow the detailed characterization of the function of the protein. Login to comment
204 As shown in Fig. 3, none of the Arg-482 mutants showed any MTX transport, similarly to the entirely inactive K86M mutant and the R482G or T mutants [6,26]. Login to comment

PMID: 15753373 [PubMed] Elkind NB et al: "Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib)."

No. Sentence Comment

46 For our studies, we have generated retrovirally transduced A431 cells, expressing various levels of the wild-type ABCG2, or a functionally inactive mutant (K86M) ABCG2 variant. Login to comment
71 Accumulation of Hoechst 33342 dye was done by using intact A431 cells without (control) or with the overexpression of wild-type ABCG2 or its inactive K86M mutant in a fluorescence spectrophotometer (Perkin-Elmer LS 50B, Perkin-Elmer/Applied Biosystems, Foster City, CA) at 350 nm (excitation)/460 nm (emission) as described (31). Login to comment

PMID: 15769853 [PubMed] Henriksen U et al: "Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2."

No. Sentence Comment

15 The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. Login to comment
16 The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. Login to comment
17 We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Login to comment
18 Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. Login to comment
20 Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. Login to comment
22 Key words: ABC transporters, ABCG2, Oligomerization, Trafficking, Multidrug resistance, Walker A mutation Summary Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2 Ulla Henriksen1 , Ulrik Gether1 and Thomas Litman2, * 1 Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, Blegdamsvej 3, University of Copenhagen, DK-2200 Copenhagen, Denmark 2 Bioinformatics Centre, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark *Author for correspondence (e-mail: tlitman@binf.ku.dk) Accepted 13 January 2005 Journal of Cell Science 118, 1417-1426 Published by The Company of Biologists 2005 doi:10.1242/jcs.01729 Research Article segments. Login to comment
28 In agreement, we find that the mutant (ABCG2-K86M) is inactive. Login to comment
36 Construction of mutation and tags The ABCG2-K86M mutation was generated by a two-generation PCR technique using the Pfu polymerase (Stratagene, La Jolla, CA). Login to comment
38 ABCG2-wt was tagged with either the MYC epitope or the HA epitope whereas ABCG2-K86M was tagged with the HA epitope. Login to comment
89 Results To obtain a loss-of-function mutant of ABCG2 we mutated the conserved Walker A lysine to methionine (K86M). Login to comment
90 The ABCG2-K86M mutant was tagged at the N-terminus with the hemagglutinin (HA) epitope whereas ABCG2-wt was tagged with a MYC epitope. Login to comment
93 The epitope tags did not alter apparent expression; however, the expression levels of the K86M mutants were somewhat lower than those observed for the wild-type constructs (Fig. 1A). Login to comment
94 The activity of ABCG2-K86M in comparison to ABCG2-wt was first assessed by measurement of ATPase activity. Login to comment
95 Whereas the ABCG2-wt membrane fraction showed dose-dependent and saturatable stimulation of ATPase activity in response to increasing concentrations of the substrate prazosin (EC50=3.5 µM; Vmax=7.6 nmol/minute/mg protein), no stimulation of the ATPase activity was detected in membranes from cells expressing ABCG2-K86M and the basal ATPase activity was comparable to that of the empty HEK293 cells (Fig. 1B). Login to comment
98 In contrast, ABCG2-K86M and ABCG2-K86M-HA displayed sensitivity comparable to that of non-transfected cells consistent with loss of function with IC50 values for mitoxantrone of 0.047 µM and 0.043 µM, respectively (Fig. 1C). Login to comment
100 These results further confirmed that ABCG2-K86M was non-functional. Login to comment
101 Cells expressing ABCG2-wt and ABCG2-wt-MYC efficiently expelled the substrate; however, this was not the case for cells expressing ABCG2-K86M or ABCG2-K86M-HA, which displayed transport activity comparable to non-transfected cells (Fig. 2). Login to comment
104 We wanted to further test this hypothesis and also analyze whether the K86M mutation altered the oligomerization properties of ABCG2. Login to comment
105 Accordingly, we coexpressed ABCG2-wt-MYC with ABCG2-K86M-HA as well as we coexpressed ABCG2-wt-MYC with ABCG2-wt tagged with HA instead of MYC. Login to comment
108 Both ABCG2-wt-HA and ABCG2-K86M-HA co-immunoprecipitated with ABCG2-wt-MYC (Fig. 3, lanes 1,2). Login to comment
111 However, we did not see the difference between the cell lines as a general phenomenon in our immunoprecipitations and in all our other experiments equal amounts of wt-HA and K86M-HA were precipitated (data not shown). Login to comment
114 Several controls were included to exclude non-specific interactions in the co-immunoprecipitation assay; cells transfected with either ABCG2-wt-MYC or ABCG2-K86M-HA showed no crossreactivity between the two tags (Fig. 3, lanes 3,4). Login to comment
119 The K86M mutation in ABCG2 results in a non-functional transporter. Login to comment
120 (A) Protein expression analyzed on isolated membrane fractions from control (empty) HEK293 cells, or HEK293 cells stably expressing ABCG2-wt, ABCG2-wt-MYC (ABCG2-wt-cmyc), ABCG2-K86M and ABCG2-K86M-HA. Login to comment
124 Vanadate-sensitive drug stimulated ATPase activity (mean±s.e.m., n=6) was measured with increasing concentrations of prazosin (0-50 µM) on ABCG2-wt, ABCG2-K86M and empty HEK293 cells. Login to comment
128 Sulforhodamine B staining was used to detect survival of ABCG2-wt, ABCG2-wt-MYC (ABCG2-wt-cmyc), ABCG2-K86M, ABCG2-K86M-HA and empty HEK293 cells. Login to comment
130 HEK293 empty ABCG2-wt-myc ABCG2-K86M ABCG2-K86M-HAABCG2-wt Fig. 2. Login to comment
131 HEK293 cells transfected with ABCG2-K86M display no transport activity. Login to comment
138 Altogether, this suggests that both ABCG2-wt and ABCG2-K86M form disulfide-bridge linked dimers. Login to comment
143 We examined this by ATPase activity measurements on isolated membrane fractions from cells coexpressing ABCG2-wt-MYC and ABCG2-K86M-HA, cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA, or cells expressing ABCG2-wt alone. Login to comment
145 We also found based on densitometry analysis of several western blots that the co-transfected cells expressed equal amounts of HA-tagged transporter (ABCG2-K86M-HA or ABCG2-wt-HA) and MYC-tagged transporter (ABCG2-wt-MYC) (Fig. 5B). Login to comment
148 However, in cells coexpressing ABCG2-wt-MYC and ABCG2-K86M-HA we found a markedly lower Vmax both compared to cells expressing ABCG2-wt alone and to cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA (EC50= 3.8±0.9 µM; Vmax=5.2±0.1 nmol/minute/mg protein; P<0.0001, unpaired t-test, Vmax compared to either ABCG2-wt alone or ABCG2-wt-MYC/ABCG2-wt-HA). Login to comment
149 The decrease in activity upon co-transfection of ABCG2-wt and ABCG2-K86M is consistent with a dominant-negative effect caused by the formation of an inactive wt/K86M complex and, accordingly, supports the fact that ABCG2 is a functional dimer. We should note, however, that based on the dominant-negative effect observed, we cannot exclude the fact that the transporters may exist and function as a higher order form than a dimer. We also investigated the subcellular localization of ABCG2-wt in comparison to ABCG2-K86M. Login to comment
150 Interestingly, immunocytochemical studies showed a distinct staining pattern of ABCG2-wt in comparison to ABCG2-K86M. Login to comment
152 In contrast, ABCG2-K86M staining was punctuate, and found almost exclusively in an intracellular compartment (Fig. 6D,E); hence, mutation of Lys86 might not only affect the functional properties of the transporter but also its cellular targeting. Login to comment
153 In order to obtain a quantitative measurement for the decrease in surface expression of the K86M mutation compared to ABCG2-wt, the cell lines were Fig. 3. Login to comment
154 ABCG2-wt and ABCG2-K86M co-immunoprecipitate. Login to comment
169 The experiments showed that the apparent surface expression of ABCG2-K86M was ~30% of that observed for the wild type consistent with our immunofluorescence data (Fig. 7A). Login to comment
170 In cells coexpressing ABCG2-wt-MYC and ABCG2-wt-HA or ABCG2-wt-MYC and ABCG2-K86M-HA we observed that the apparent surface expression in both cases was 30-40% higher than in cells only expressing ABCG2-wt-MYC. Login to comment
172 This was supported by immunostaining cells expressing K86M alone in comparison to cells expressing both K86M-HA and wt-MYC. Login to comment
173 As shown in Fig. 7B the vast majority of anti-HA staining was found intracellularly in cells expressing only K86M-HA whereas in the cells expressing K86M-HA together with wt-MYC we observed increased plasma membrane anti-HA staining. Login to comment
174 To determine the subcellular compartment in which ABCG2-K86M was localized we performed a series of co-stainings using the anti-ABCG2 antibody together with markers for different cellular compartments. Login to comment
175 We found similar staining patterns for ABCG2-K86M and anti-calnexin, a marker for the endoplasmic reticulum (ER) (Fig. 8B). Login to comment
177 Altogether, we conclude that ABCG2-K86M is mainly localized to the ER although a small percentage is localized to the plasma membrane. Login to comment
178 To verify our localization results we analyzed the glycosylation state of ABCG2-wt and ABCG2-K86M. Login to comment
196 These data imply that ABCG2-K86M is processed beyond the ER and possibly to cis-Golgi from where it is retrieved again to the ER where it is mainly detected. Login to comment
198 Furthermore, we show that mutation to methionine of the highly conserved lysine in Walker A (K86M) not only results in a non-functional transporter but also has a profound effect on targeting of the transporter to the cell surface. Login to comment
203 In ABCG2, it has been demonstrated by expression in Sf-9 insect cells that K86M is functionally inactive but still capable of binding ATP (Ozvegy et al., 2002). Login to comment
204 Accordingly, we chose K86M to address whether ABCG2 is a functional oligomer and established co-transfected stable HEK293 cell lines expressing the wild type alone, K86M alone or wild type and K86M half-transporter together. Login to comment
205 Analysis of the cotransfected cells with respect to ATPase activity suggested a dominant-negative activity of the K86M mutant on wild-type activity, i.e. membranes from cells coexpressing wt-MYC and K86M-HA displayed less ATPase activity as compared to cells expressing the wild type alone and only about 40% of the ATPase activity was observed in cells expressing both wt-HA and wt-MYC (Fig. 5). Login to comment
207 ABCG2-K86M is localized in an intracellular compartment. Login to comment
211 (A) ABCG2-wt, (B) ABCG2-wt-HA, (C) ABCG2-wt-MYC (ABCG2-wt-cmyc), (D) ABCG2-K86M and (E) ABCG2-K86M-HA. Login to comment
213 ABCG2-K86M shows a markedly reduced surface expression. Login to comment
219 (B) HEK293 cells stably expressing ABCG2-K86M or ABCG2-wt-MYC + ABCG2-K86M-HA were analyzed by immunocytochemistry to detect localization of the transporter. Login to comment
223 One possible explanation for this apparent discrepancy is that we have used stably transfected pool clones and accordingly cannot assume that the wild type and K86M are expressed in exactly equal amount in all cells. Login to comment
224 It is also possible that because the wild type and K86M preferentially are targeted to the ER and cell surface, respectively, we might not obtain the ideal distribution of 50% wt/K86M dimers, 25% wt/wt dimers and 25% K86M/ K86M. Login to comment
225 The presence of a dominant-negative effect supports the idea that the inactive K86M mutant is capable of oligomerizing with the wild type resulting in a nonfunctional wt/K86M transporter protein complex and, thus, that at least two functional NBDs are necessary for proper ATP hydrolysis. Login to comment
231 The ABCG2-K86M mutant resides in the ER. Login to comment
238 ABCG2-K86M is glycosylated to the same degree as ABCG2-wt. Login to comment
240 Total cell lysates of ABCG2-wt or ABCG2-K86M were treated with denaturation buffer for 10 minutes at 100°C. Login to comment
248 We also assessed the oligomerization properties of ABCG2-wt and ABCG2-K86M by co-immunoprecipitation. Login to comment
259 Surprisingly, we observed a striking difference between the localization of ABCG2-wt (plasma membrane) and ABCG2-K86M (ER). Login to comment
261 A previous study on ABCG2-K86M suggested normal surface expression in Sf9 insect cells (Ozvegy et al., 2002). Login to comment
263 Immunocytochemical staining suggests that the K86M mutant resides in the ER, either because of retention or retrieval of the transporter to this compartment. Login to comment
264 In this context it is interesting to note that western blot analysis comparing the wild type and K86M showed that they both migrate identically in the gel. Login to comment
266 The glycosylation was, however, insensitive to Endo H indicating that both ABCG2-wt and ABCG2-K86M has been processed beyond the ER. Login to comment
267 This supports a scenario in which the K86M mutant is processed to the Golgi or the intermediary compartment and subsequently retrieved to the ER as part of a putative quality control mechanism. Login to comment
268 The available structural data on ABC transporters gives rise to speculation regarding how the K86M mutation affects surface targeting. Login to comment
271 As it was previously shown that the ABCG2-K86M mutation can still bind but not hydrolyze ATP (Ozvegy et al., 2002), a conceivable scenario might be that hydrolysis of ATP facilitates a closed structure of the NBDs; hence, a transporter unable to hydrolyse ATP is likely to have a markedly looser structure. Login to comment
272 Since the K86M mutation directly affects the ATP binding site, the NBD dimerization interface could be affected leading to impaired surface targeting followed by retrieval to ER. Login to comment
273 We still observe dimerization of the transporter when mutating K86M, suggesting that dimer formation is likely to be dependent on the transmembrane domains as well as on disulfide bridges formed between cysteines present in the predicted extracellular loops. Login to comment

PMID: 15838659 [PubMed] Morisaki K et al: "Single nucleotide polymorphisms modify the transporter activity of ABCG2."

No. Sentence Comment

76 Cells were also infected with mutant R482G ABCG2 encoding a K86M mutation that has been shown to abolish the function of the resulting protein [34]. Login to comment
163 To examine whether the nonsynonymous SNPs in ABCG2 affect the transport of this compound, Hoechst 33342 dye transport was measured in intact Sf9 cells expressing wild-type, V12M, Q141K, or D620N ABCG2, as well as the nonfunctional mutant, R482G/K86M. Login to comment
167 In Sf9 cells expressing the R482G/K86M ATP binding-site mutant, Hoechst 33342 uptake was comparable to the level of Hoechst 33342 accumulation found in the Ko143-inhibited cells. Login to comment

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

242 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 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: 16107343 [PubMed] Henriksen U et al: "Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2."

No. Sentence Comment

25 However, our previous data also shows that dimerization of the transporter is not dependent on functional nucleotide binding domains, as an inactivating mutation (K86M) in the Walker A motif did not alter the ability of ABCG2 to form dimers (24). Login to comment

PMID: 16402910 [PubMed] Krishnamurthy P et al: "Role of ABCG2/BCRP in biology and medicine."

No. Sentence Comment

159 Moreover, a nonfunctional ABCG2 mutant (bearing a K86M mutation in the Walker A NBD) acted as an effective dominant-negative inhibitor of ABCG2 when cotransfected with a nonmutated ABCG2 (83, 88). Login to comment

PMID: 16618113 [PubMed] Polgar O et al: "Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization."

No. Sentence Comment

86 Generation of Sf9 Cells Expressing the Wild Type or the K86M or G553L Mutant. Login to comment
87 Generation of transfer vectors containing wt ABCG2 or K86M has been described previously (26, 27). Login to comment
101 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 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
148 The ATPase activity was comparable to that of the nonfunctional K86M mutant (26) (Figure 6B). Login to comment
159 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
161 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
173 (B) The G553L mutant displays basal ATPase activity similar to that of the nonfunctional K86M mutant in Sf9 membranes. Login to comment
203 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 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 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
222 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 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

PMID: 17015488 [PubMed] Sarkadi B et al: "Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system."

No. Sentence Comment

1084 Clearly, a nonfunctional mutant (K86M) ABCG2 variant induces a dominant negative effect, that is, a nonfunctional dimer formation suppresses the activity of the wild-type protein (93, 166). Login to comment

PMID: 17027309 [PubMed] Li YF et al: "Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study."

No. Sentence Comment

109 Mutations T82A, K86M, and K86I [42,43] are part of the Walker A motif; all lead to loss of transport activity. Login to comment
111 The K86M or the K86I mutation leads to loss of interaction with the tri-phosphate group of the bound ATP, and thus inactivates the enzyme. Login to comment
174 Subsequently, a symmetric dimer Y.-F Li et al. / Journal of Molecular Graphics and Modelling 25 (2007) 837-851844 Table 4 Locations of mutations as predicted by the ABCG2 model and functional correlation Mutation Position in ABCG2 Phenotype Reference V12M N-terminal Membrane localization, SNP, and somewhat lower expression and lower resistance [22] S25Pa N-terminal Low drug resistance for the cell line due to lower expression at cell surface [42] T82Aa NBD, Walker A Low drug resistance for the cell line due to lower expression at cell surface [42] K86M NBD, Walker A No expression at cell surface, retained in the Golgi [43] K86I NBD, Walker A No expressed at cell surface [43] Q141K NBD SNP with lower protein expression and low drug resistance [22,23] T237V NBD Fully functional b I239K,R NBD Loss of expression may be due to structural disruption b R309G Linkerc Low drug resistance [42] D315-6 Linker Deletion mutant for A315 and T316. Login to comment

PMID: 17092765 [PubMed] Assaraf YG et al: "The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis."

No. Sentence Comment

196 First, co-introduction of both a wild type as well as a catalytically null mutant ABCG2 cDNA bearing a K86M mutation in the consensus Walker A motif in the NBF resulted in a negative-dominant effect thereby abolishing ABCG2 function (Ozvegy et al., 2001, 2002). Login to comment

PMID: 17346156 [PubMed] Xu J et al: "Human multidrug transporter ABCG2, a target for sensitizing drug resistance in cancer chemotherapy."

No. Sentence Comment

31 Moreover, the non-functional ABCG2 mutant (bearing a K86M mutation in the Walker A NBD) acted as an effective dominant-negative inhibitor of ABCG2 when co-expressed with a wild type ABCG2 [14, 18]. Login to comment

PMID: 17537873 [PubMed] Glavinas H et al: "ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions."

No. Sentence Comment

45 Human membrane vesicle preparations containing ABCG2 (MXR-M) and control human membrane preparations (M-CTRL), as well as insect cell membranes containing the human transporter (MXR-Sf9) and control insect membranes (beta-gal-Sf9-CTRL, MXR-K86M-Sf9-CTRL), were obtained from SOLVO Biotechnology (Budapest, Hungary, http://www. Login to comment
47 The insect membrane vesicle preparations were obtained using recombinant baculoviruses encoding wild-type human ABCG2, inactive ABCG2-K86M mutant (carrying a mutation at a crucial position of the catalytic center of ATP binding and cleavage), and beta-galactosidase (Ozvegy et al., 2001, 2002). Login to comment
102 Sf9 preparations containing the wild-type or the defective (K86M) transporter (MXR-Sf9, lane 1; MXR-K86M-Sf9-CTRL, lane 2) displayed a strong band with apparent molecular mass of 55 to 60 kDa. Login to comment
110 The transport of [3 H]methotrexate could not be observed in Sf9 membranes containing the K86M- FIG. 2. Login to comment
111 Vanadate-sensitive ATPase activity of MXR-Sf9 (A, C) and MXR-M (B, D) and defMXR-K86M-Sf9 (E) and M-CTRL (F) preparations in the presence of ABCG2 substrates and inhibitors at different concentrations. Membranes containing 20 ␮g of total protein were incubated at 37°C for 40 min in the presence of different concentrations of test compounds. Login to comment
125 Sf9 membranes expressing the defective (MXR-K86M-Sf9-CTRL) version of ABCG2 show similar baseline vanadate-sensitive ATPase activity, which was not modulated by any of the substrates tested (Fig. 2E). Login to comment
181 The vanadate-sensitive ATPase activity of MXR-Sf9 membranes in the presence of Ko143 or Ko134 and the vanadate-sensitive ATPase activity present of membranes containing the defective transporter (MXR-K86M-Sf9-CTRL) show that the Sf9 preparations contain some (ϳ10 nmol Pi/mg/min) non-ABCG2-related vanadate-sensitive FIG. 5. Login to comment

PMID: 18089722 [PubMed] Wu CP et al: "Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter."

No. Sentence Comment

200 Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2. Login to comment

PMID: 18182157 [PubMed] Orban TI et al: "Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter."

No. Sentence Comment

29 As a control, K86M catalytic site mutant was also created for the tagged proteins [10]. Login to comment

PMID: 18249138 [PubMed] Hazai E et al: "Homology modeling of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

245 However, in our model, R482 cannot form interaction with rhodamine, but L484 is in interacting distance Table 3 Mutations on BCRP and their effect on its function Mutation Effect/results Reference V12M Did not effect Hemato and MTX transport Tamura et al. (2006) G51C Did not effect Hemato and MTX transport Tamura et al. (2006) K86M Inactivates transporter (dominant negative effect on ATPase activity); alters subcellular distribution Henriksen et al. (2005a) K86M Transporter inactive, but still able to bind ATP Ozvegy et al. (2002) Q126stop Defective porphyrin transport Tamura et al. (2006) Q141K Did not effect Hemato and MTX transport Tamura et al. (2006) T153M Did not effect Hemato and MTX transport Tamura et al. (2006) Q166E Did not effect Hemato and MTX transport Tamura et al. (2006) I206L Did not effect Hemato and MTX transport Tamura et al. (2006) F208S Defective porphyrin transport Tamura et al. (2006) S248P Defective porphyrin transport Tamura et al. (2006) E334stop Defective porphyrin transport Tamura et al. (2006) F431L Effects MTX transport Tamura et al. (2006) S441N Defective porphyrin transport Tamura et al. (2006) E446-mutants No drug resistance Miwa et al. (2003) R482G, R482T Effects MTX transport Tamura et al. (2006) R482T Substrate drug transport and inhibitor efficiency is not mediated by changes in drug-binding Pozza et al. (2006) R482G, R482T Substitution influence the substrate specificity of the transporter Ozvegy et al. (2002) R482G, R482T Altered substrate specificity Honjo et al. (2001) R482G Methotrexate not transported Chen et al. (2003b) Mitomo et al. (2003) R482G Resistance to hydrophilic antifolates in vitro, G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates Shafran et al., (2005) R482G Three distinct drug, binding sites Clark et al. (2006) R482G Altered substrate specificity, granulocyte maturation uneffected Ujhelly et al. (2003) R482 mutants Higher resistance to mitoxantrone and doxorubicin than wt Miwa et al. (2003) R482X Affects substrate transport and ATP hydrolysis but not substrate binding Ejendal et al. (2006) F489L Impaired porphyrin transport Tamura et al. (2006) G553L; G553E Impaired trafficing, expression, and N-linked glycosylation Polgar et al. (2006) L554P Dominant negative effect on drug sensitivity Kage et al. (2002) N557D Resistance to MTX, but decreased transport of SN-38; N557E no change in transport compared to wt Miwa et al. (2003) F571I Did not effect Hemato and MTX transport Tamura et al. (2006) N590Y Did not effect Hemato and MTX transport Tamura et al. (2006) C592A Impaired function and expression Henriksen et al. (2005b) C592A/C608A Restored plasma mb expression; MTX transport normal, BODIPY-prazosin impaired Henriksen et al. (2005b) C603A Disulfide bridge; no functional or membrane targeting change Henriksen et al. (2005b) C608A Impaired function and expression Henriksen et al. (2005b) D620N Did not effect Hemato and MTX transport Tamura et al. (2006) H630X No change in transport Miwa et al. (2003) Cand N-terminal truncated Impaired trafficing Takada et al. (2005) with the ligand. Login to comment

PMID: 18370855 [PubMed] Polgar O et al: "ABCG2: structure, function and role in drug response."

No. Sentence Comment

378 Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2. Login to comment

PMID: 18397960 [PubMed] Kis E et al: "Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance."

No. Sentence Comment

39 The insect membrane vesicle preparations were obtained using recombinant baculoviruses encoding wild-type human BCRP, defective BCRP-K86M mutant (carrying a mutation at a crucial position of the catalytic center of ATP binding and cleavage) and MDR1.21 Sf9 cells were cultured and infected with recombinant baculovirus stocks as described earlier. Login to comment

PMID: 19135105 [PubMed] Hegedus C et al: "Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays."

No. Sentence Comment

1174 After transient transfection of cells with plasmids expressing GFP-ABCG2 or the nonfunctional GFP-ABCG2 K86M mutant protein, the fluorescent fusion species were found to be well-suited for rapid flow cytometry or fluorescence microscopy applications [102]. Login to comment
1178 Using a specific inhibitor, either Ko143 or FTC [10], and comparing the results with those of the GFP-tagged K86M species, the transport characteristics of the applied fluorescent drug can be rapidly and reliably assessed. Login to comment

PMID: 19406100 [PubMed] Polgar O et al: "Arginine 383 is a crucial residue in ABCG2 biogenesis."

No. Sentence Comment

169 (C) Basal ATPase activity of the wild-type, a 1:5 dilution of the wild-type, the R383A mutant, and the non-functional K86M mutant in Sf9 membranes. 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

134 However, in considering the results published by Henriksen et al., i.e., coexpression of wt ABCG2 with K86M-mutated ABCG2 exerted ~50% of wt ABCG2 ATPase activity (54), the above result might be interpreted as that one of the two ATPs bound to the heterodimer of YFP/ABCG2 and E211Q could be hydrolyzed. Login to comment
175 This is consistent with Henriksen`s result that coexpression of wt ABCG2 with K86M-mutated ABCG2 exerted ~50% of wt ABCG2 ATPase activity (54). Login to 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: 20665261 [PubMed] Pozza A et al: "Insect cell versus bacterial overexpressed membrane proteins: an example, the human ABCG2 transporter."

No. Sentence Comment

364 The basal ATPase activity was determined on inverted membrane vesicles from control insect cells expressing b-galactosidase or inactive K86M ABCG2. Login to comment
361 The basal ATPase activity was determined on inverted membrane vesicles from control insect cells expressing b-galactosidase or inactive K86M ABCG2. Login to comment

PMID: 16640501 [PubMed] Su Y et al: "Drug delivery across the blood-brain barrier: why is it difficult? how to measure and improve it?"

No. Sentence Comment

378 HENRIKSEN U, GETHER U, LITMAN T: Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2. Login to comment

PMID: 15240127 [PubMed] Cserepes J et al: "Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization."

No. Sentence Comment

87 For comparison, we chose to express the glycine variant, ABCG2R482G (and its catalytic site mutant ABCG2R482G; K86M), which is well characterized and its ATPase activity is stimulated by Rhodamine123 [17] as we found for ABCG1 (see below [2]). Login to comment

PMID: 24312054 [PubMed] Gauthier C et al: "ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity."

No. Sentence Comment

52 MEMBRANE PREPARATION For obtaining membrane vesicles insect cells were infected with recombinant baculoviruses containing the cDNA of wtABCG2 or ABCG2-K86M (Ozvegy-Laczka et al., 2005) or of ABCC1 (Bakos 3 - #3 Gauthier et al. ABCG2 inability to transport glutathione et al.,1996). Login to comment
114 There was a low level of GSH accumulation in the presence of ABCG2 observed without ATP, which was also observed in the presence of the selective ABCG2 inhibitor Ko143 (Allen et al., 2002), or when the catalytically inactive K86M ABCG2 mutant was expressed. Login to comment
134 ATP-dependent transport of 3H-methotrexate in 2 mM cholesterol-loaded insect-cell membranes expressing ABCG2 (either wild-type or the inactive K86M mutant) was measured for 10 min at 37e6;C. Login to comment

PMID: 24384916 [PubMed] Telbisz A et al: "Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites."

No. Sentence Comment

48 Generation of the baculovirus transfer vector (pAcUW21-L) harboring the cDNA for wild-type (wt) ABCG2 or the R482 and K86M mutants was described previously (Ozvegy et al., 2002; Ozvegy-Laczka et al., 2005). Login to comment
187 However, the most pronounced effect of CA is observed in cholesterol-loaded membranes (Fig. 4B, right columns): baseline ATPase activity is strongly reduced (almost to the level of Sf9 membranes expressing the inactive ABCG2-K86M mutant; see Fig. 2B), while drug-stimulated ATPase activity is unchanged. Login to comment
299 Moreover, we observed that when membranes were loaded with cholesterol, CA decreased the baseline ATP hydrolysis down to the background level, that is, to ATP hydrolysis in membranes expressing the inactive mutant ABCG2-K86M (see Fig. 2B). Login to comment

PMID: 25036722 [PubMed] Szafraniec MJ et al: "Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

196 Studies on Lys86 Met-BCRP, catalytically inactive, but able to bind ATP, revealed that this mutation has no influence on the oligomerization properties of the transporter but prevents the stimulation of ATPase activity by prazosin. Login to comment
197 The ability of cells stably transfected with Lys86 Met-BCRP to transport mitoxantrone was comparable to that of non-transfected cells. Login to comment
198 A co-transfection of HEK293 with both WT and Lys86 Met proteins resulted in a 60% loss of ATPase activity. Login to comment
199 A possible explanation of this is that the Lys86 Met protein oligomerizes with the WT protein creating a non-functional complex, as two active NBDs are necessary for ATP hydrolysis (Henriksen et al., 2005b). Login to comment
209 Position Type of mutation Effect on the transporter References NBD Lys 86 Met (i) No stimulation of the ATPase activity by prazosin; (ii) no influence on the transport of mitoxantrone Henriksen et al. (2005b) Glu 126 stop, Phe 208 Ser, Ser 248 Phe, Glu 334 stop Inability to transport hematoporphyrin Tamura et al. (2006) Glu 211 Gln Complete abolishment of the ATPase activity and methotrexate transport Hou et al. (2009) Pro 392 Ala Significant reduction in the efflux activity of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Ni et al. (2011) TM1 Gly 406 Ala Gly 410 Ala No influence on the activity of the transporter Polgar et al. (2004) Gly 406 Leu Gly 410 Leu (i) Loss of the ability to transport rhodamine123; (ii) impaired transport of mitoxantrone, Pheide and BODIPY-prazosin Polgar et al. (2004) Extracellular loop 1 Phe 431 Leu (i) Loss of the ability to transport methotrexate; (ii) 10% level of hematoporphyrin transport compared to the WT protein Tamura et al. (2006) Ser 441 Asn Inability to transport hematoporphyrin Tamura et al. (2006) Ser 441 Asn Loss of the ability to transport methotrexate Tamura et al. (2006) TM2 Lys 452 Ala His 457 Ala Increase in transport of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Cai et al. (2010) Lys 453 Ala Arg 465 Ala Decrease in transport of mitoxantrone, BODIPY-prazosin, Hoechst 33342, doxorubicin, SN-38 and rhodamine 123 Cai et al. (2010) TM3 Arg 482 Gly Arg 482 Thr (i) No change in the inhibitory activity of lapatinib; (ii) about two times greater inhibition by ritonavir, saquinavir and nalfinavir than in the WT variant; (iii) gaining the ability to transport rhodamine123 and doxorubicin; (iv) no influence on the transport of mitoxantrone; (v) loss of the ability to transport methotrexate Dai et al. (2008), Gupta et al. (2004), Honjo et al. (2001), Mitomo et al. (2003) Arg 482 Thr (i) Lower IC 50 of cyclosporine A for mutant than for WT variant; (ii) lower elacridar inhibition potency Xia et al. (2007) Arg 482 Lys Complete loss of transport activity Ejendal et al. (2006) Phe 489 Leu Impaired transport of porphyrins, no transport of methotrexate Tamura et al. (2006) Extracellular loop 3 Asn 590 Tyr Over twice reduced transport of mitoxantrone, topotecan, daunorubicin and rhodamine 123 Vethanayagam et al. (2005) Cys 592 Ala/Cys 608 Ala (i) Transport of mitoxantrone almost unchanged; (ii) transport of BODIPY-prazosin significantly impaired Henriksen et al. (2005a) Extracellular loop 3 Cys 603 Ser Cys 592 Ser/Cys 608 Ser Cys 592 Ser/Cys 603 Ser/Cys 608 Ser Diminished susceptibility to the inhibitory activity of fumitremorgin C Shigeta et al. (2010) Cys-less Arg 482 Gly-BCRP Complete loss of the ability to efflux mitoxantrone Liu et al. (2008b) The positions of the amino acid residues refer to the topological model of BCRP proposed by Wang et al. (2009). Login to comment

PMID: 25064323 [PubMed] Krzyzanowski D et al: "Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress."

No. Sentence Comment

155 Membrane vesicles of Sf9 insect cells, which were infected with recombinant baculoviruses containing the cDNA of wtABCG2 or ABCG2-K86M (inactive), showed no differences in ATP-dependent [3 H]methotrexate and [3 H]GSH uptake. Login to comment

PMID: 25445676 [PubMed] Gal Z et al: "Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter."

No. Sentence Comment

45 The generation of the vector construct with the ABCG2-K86M mutant was described earlier [26]. Login to comment