ABCMdb

ABCG2 p.Lys86Met

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

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[hide] Characterization of drug transport, ATP hydrolysis... J Biol Chem. 2002 Dec 13;277(50):47980-90. Epub 2002 Oct 8. Ozvegy C, Varadi A, Sarkadi B
Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation.
J Biol Chem. 2002 Dec 13;277(50):47980-90. Epub 2002 Oct 8., 2002-12-13 [PMID:12374800]

Abstract [show]
The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. 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. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.

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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

[hide] Multidrug resistance mediated by the breast cancer... Oncogene. 2003 Oct 20;22(47):7340-58. Doyle LA, Ross DD
Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2).
Oncogene. 2003 Oct 20;22(47):7340-58., 2003-10-20 [PMID:14576842]

Abstract [show]
Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.

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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

[hide] Functional characterization of human breast cancer... Mol Pharmacol. 2003 Dec;64(6):1452-62. Nakanishi T, Doyle LA, Hassel B, Wei Y, Bauer KS, Wu S, Pumplin DW, Fang HB, Ross DD
Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis.
Mol Pharmacol. 2003 Dec;64(6):1452-62., [PMID:14645676]

Abstract [show]
To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.

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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

[hide] Single nucleotide polymorphisms result in impaired... Int J Cancer. 2004 Mar 20;109(2):238-46. Mizuarai S, Aozasa N, Kotani H
Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2.
Int J Cancer. 2004 Mar 20;109(2):238-46., 2004-03-20 [PMID:14750175]

Abstract [show]
ABCG2/MXR/ABCP1/BCRP is a member of the ATP-binding cassette membrane transporter, which consists of six transmembrane regions and one ATP-binding cassette. The transporter is known to be involved in the efflux of various anticancer compounds such as mitoxantrone, doxorubicin and topoisomerase I inhibitor. In this study, we analyzed the effects of polymorphisms in ABCG2, V12M and Q141K on transporter function. When polarized LLC-PK1 cells were transfected with variant ABCG2, drug-resistance to topoisomerase I inhibitor of cells expressing V12M or Q141K was less than 1/10 that of wild-type ABCG2 transfected cells, and was accompanied by increased drug accumulation and decreased drug efflux in the variant ABCG2-expressing cells. We further elucidated the molecular mechanisms of the transport dysfunction by investigating membrane localization and ATPase activity. Confocal microscopic analysis revealed that apical plasma membrane localization of V12M was disturbed, while the localization of wild-type transporters occurred specifically in the apical plasma membrane of polarized LLC-PK1 cells. Also, ATPase activities measured in the membrane of SF9 cells infected with variant ABCG2 showed that Q141K decreased activity by 1.3 below that of wild-type ABCG2. In addition, kinetic analysis of ATPase activity showed that the K(m) value in Q141K was 1.4-fold higher than that of wild-type ABCG2. These results indicated that naturally occurring SNPs alter transport functions of ABCG2 transporter and analysis of SNPs in ABCG2 may hold great importance in understanding the response/metabolism of chemotherapy compounds that act as substrates for ABCG2.

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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

[hide] Function-dependent conformational changes of the A... J Biol Chem. 2005 Feb 11;280(6):4219-27. Epub 2004 Nov 22. Ozvegy-Laczka C, Varady G, Koblos G, Ujhelly O, Cervenak J, Schuetz JD, Sorrentino BP, Koomen GJ, Varadi A, Nemet K, Sarkadi B
Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface.
J Biol Chem. 2005 Feb 11;280(6):4219-27. Epub 2004 Nov 22., 2005-02-11 [PMID:15557326]

Abstract [show]
The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. 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. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.

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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

[hide] Single amino acid (482) variants of the ABCG2 mult... Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63. Ozvegy-Laczka C, Koblos G, Sarkadi B, Varadi A
Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition.
Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63., 2005-02-01 [PMID:15670731]

Abstract [show]
The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

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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

[hide] Multidrug transporter ABCG2 prevents tumor cell de... Cancer Res. 2005 Mar 1;65(5):1770-7. Elkind NB, Szentpetery Z, Apati A, Ozvegy-Laczka C, Varady G, Ujhelly O, Szabo K, Homolya L, Varadi A, Buday L, Keri G, Nemet K, Sarkadi B
Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).
Cancer Res. 2005 Mar 1;65(5):1770-7., 2005-03-01 [PMID:15753373]

Abstract [show]
Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.

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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

[hide] Effect of Walker A mutation (K86M) on oligomerizat... J Cell Sci. 2005 Apr 1;118(Pt 7):1417-26. Epub 2005 Mar 15. Henriksen U, Gether U, Litman T
Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2.
J Cell Sci. 2005 Apr 1;118(Pt 7):1417-26. Epub 2005 Mar 15., 2005-04-01 [PMID:15769853]

Abstract [show]
The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. 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. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. 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. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.

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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

[hide] Single nucleotide polymorphisms modify the transpo... Cancer Chemother Pharmacol. 2005 Aug;56(2):161-72. Epub 2005 Apr 19. Morisaki K, Robey RW, Ozvegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B, Bates SE
Single nucleotide polymorphisms modify the transporter activity of ABCG2.
Cancer Chemother Pharmacol. 2005 Aug;56(2):161-72. Epub 2005 Apr 19., [PMID:15838659]

Abstract [show]
Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P = 0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

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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

[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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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

[hide] Identification of intra- and intermolecular disulf... J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17. Henriksen U, Fog JU, Litman T, Gether U
Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2.
J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17., 2005-11-04 [PMID:16107343]

Abstract [show]
ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

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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

[hide] Role of ABCG2/BCRP in biology and medicine. Annu Rev Pharmacol Toxicol. 2006;46:381-410. Krishnamurthy P, Schuetz JD
Role of ABCG2/BCRP in biology and medicine.
Annu Rev Pharmacol Toxicol. 2006;46:381-410., [PMID:16402910]

Abstract [show]
The protein variously named ABCG2/BCRP/MXR/ABCP is a recently described ATP-binding cassette (ABC) transporter originally identified by its ability to confer drug resistance that is independent of Mrp1 (multidrug-resistance protein 1) and Pgp (P-glycoprotein). Unlike Mrp1 and Pgp, ABCG2 is a half-transporter that must homodimerize to acquire transport activity. ABCG2 is found in a variety of stem cells and may protect them from exogenous and endogenous toxins. ABCG2 expression is upregulated under low-oxygen conditions, consistent with its high expression in tissues exposed to low-oxygen environments. ABCG2 interacts with heme and other porphyrins and protects cells and/or tissues from protoporphyrin accumulation under hypoxic conditions. Individuals who carry ABCG2 alleles that have impaired function may be more susceptible to porphyrin-induced toxicity. Abcg2 knock-out models have allowed in vivo studies of Abcg2 function in host and cellular defense. In combination with immunohistochemical analyses, these studies have revealed how ABCG2 influences the absorption, distribution, and excretion of drugs and cytotoxins.

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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

[hide] Mutational studies of G553 in TM5 of ABCG2: a resi... Biochemistry. 2006 Apr 25;45(16):5251-60. 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 [PMID:16618113]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a three-amino acid sequence in transmembrane segment 5 (residues 552-554). Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedly reduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylation was suggested by a reduction in molecular mass compared to that of the 72 kDa wild-type ABCG2. Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein. 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. Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.

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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

[hide] Human multidrug resistance ABCB and ABCG transport... Physiol Rev. 2006 Oct;86(4):1179-236. Sarkadi B, Homolya L, Szakacs G, Varadi A
Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.
Physiol Rev. 2006 Oct;86(4):1179-236., [PMID:17015488]

Abstract [show]
In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

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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

[hide] Towards understanding the mechanism of action of t... J Mol Graph Model. 2007 Mar;25(6):837-51. Epub 2006 Aug 30. Li YF, Polgar O, Okada M, Esser L, Bates SE, Xia D
Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study.
J Mol Graph Model. 2007 Mar;25(6):837-51. Epub 2006 Aug 30., [PMID:17027309]

Abstract [show]
The ATP-binding cassette protein ABCG2 is a member of a broad family of ABC transporters with potential clinical importance as a mediator of multidrug resistance. We carried out a homology and knowledge-based, and mutationally improved molecular modeling study to establish a much needed structural framework for the protein, which could serve as guidance for further genetic, biochemical, and structural analyses. Based on homology with known structures of both full-length and nucleotide-binding domains (NBD) of ABC transporters and structural knowledge of integral membrane proteins, an initial model of ABCG2 was established. Subsequent refinement to conform to the lipophilic index distributions in the transmembrane domain (TMD) and to the results of site-directed mutagenesis experiments led to an improved model. The complete ABCG2 model consists of two identical subunits facing each other in a closed conformation. The dimeric interface in the nucleotide-binding domain (NBD) involves a characteristic nucleotide sandwich and the interface in the TMD consists of the TM helices 1-3 of one subunit and the helices 5 and 6 of the other. The interface between the NBD and the TMD is bridged by the conserved structural motif between TM2 and TM3, the intracellular domain 1 (ICD1), and the terminal beta-strand (S6) of the central beta-sheet in the NBD. The apparent flexibility of the ICD1 may play a role in transmitting conformational changes from the NBD to the TMD or from the TMD to the NBD.

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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

[hide] The role of multidrug resistance efflux transporte... Drug Resist Updat. 2006 Aug-Oct;9(4-5):227-46. Epub 2006 Nov 7. Assaraf YG
The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis.
Drug Resist Updat. 2006 Aug-Oct;9(4-5):227-46. Epub 2006 Nov 7., [PMID:17092765]

Abstract [show]
Members of the ATP-binding cassette (ABC) transporters including P-glycoprotein (Pgp/ABCB1), multidrug resistance proteins (MRPs/ABCC) as well as breast cancer resistance protein (BCRP/ABCG2) function as ATP-dependent drug efflux transporters, which form a unique defense network against multiple chemotherapeutic drugs and cellular toxins. Among antitumor agents is the important group of folic acid antimetabolites known as antifolates. Antifolates such as methotrexate (MTX), pemetrexed and raltitrexed exert their cytotoxic activity via potent inhibition of folate-dependent enzymes essential for purine and pyrimidine nucleotide biosynthesis and thereby block DNA replication. Overexpression of MRPs and BCRP confers resistance upon malignant cells to various hydrophilic and lipophilic antifolates. Apart from their central role in mediating resistance to antifolates and other anticancer drugs, MRPs and BCRP have been recently shown to transport naturally occurring reduced folates. This was inferred from various complementary systems as follows: (a) Cell-free systems including ATP-dependent uptake of radiolabeled folate/MTX into purified inside-out membrane vesicles from stable transfectants and/or cells overexpressing these transporters, (b) Decreased accumulation of radiolabeled folate/MTX in cultured tumor cells overexpressing these transporters, as well as (c) In vivo rodent models such as Eisi hyperbillirubinemic rats (EHBR) that hereditarily lack MRP2 in their canalicular membrane and thereby display a bile that is highly deficient in various reduced folate cofactors and MTX, when compared with wild type Sprague-Dawley (SD) rats. In all cases, these folate/antifolate transporters functioned as high capacity, low affinity ATP-driven exporters. While the mechanism of cellular retention of (anti)folates is mediated via (anti)folylpolyglutamylation, certain efflux transporters including MRP5 (ABCC5) and BCRP were shown to transport both mono-, di- as well as triglutamate derivatives of MTX and folic acid. Furthermore, overexpression of MRPs and BCRP has been shown to result in decreased cellular folate pools, whereas loss of ABC transporter expression brought about a significant expansion in the intracellular reduced folate pool. The latter finding has important implications to antifolate-based chemotherapy as an augmented cellular folate pool results in a significant level of resistance to certain antifolates. Hence, the aims of the present review are: (a) To summarize and discuss the cumulative evidence supporting a functional role for various multidrug resistance efflux transporters of the ABC superfamily which mediate resistance to hydrophilic and lipophilic antifolates, (b) To describe and evaluate the recent data suggesting a role for these efflux transporters in regulation of cellular folate homeostasis under folate replete and deplete conditions. Furthermore, novel developments and future perspectives regarding the identification of novel antifolate target proteins and mechanisms of action, as well as rationally designed emerging drug combinations containing antifolates along with receptor tyrosine kinase inhibitors are being discussed.

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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

[hide] Human multidrug transporter ABCG2, a target for se... Curr Med Chem. 2007;14(6):689-701. Xu J, Peng H, Zhang JT
Human multidrug transporter ABCG2, a target for sensitizing drug resistance in cancer chemotherapy.
Curr Med Chem. 2007;14(6):689-701., [PMID:17346156]

Abstract [show]
Human ABCG2, a member of the ATP-binding cassette transporter superfamily which transports a wide variety of substrates, is highly expressed in placental syncytiotrophoblasts, in the canalicular membranes of liver, in the apical membrane of the small intestine epithelium, and at the luminal surface of the endothelial cells of human brain micro vessels. This strategic tissue localization indicates that ABCG2 plays an important role in absorption, distribution, and elimination of xenobiotics and drugs. High ABCG2 expression has also been detected in many hematological malignancies and solid tumors, indicating that ABCG2 is likely responsible also for the multidrug resistance in cancer chemotherapy. Indeed, ABCG2 can actively transport structurally diverse conjugated- or unconjugated-organic molecules and various anticancer drugs. Many chemo-sensitizing agents have been discovered, which can be developed for increasing drug adsorption and reversing drug resistance in cancer chemotherapy by inhibiting ABCG2 function or expression. This review summarizes current knowledge on ABCG2, its relevance to multidrug resistance and drug disposition, and its ever-growing numbers of substrates and inhibitors.

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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

[hide] ABCG2 (breast cancer resistance protein/mitoxantro... Drug Metab Dispos. 2007 Sep;35(9):1533-42. Epub 2007 May 30. Glavinas H, Kis E, Pal A, Kovacs R, Jani M, Vagi E, Molnar E, Bansaghi S, Kele Z, Janaky T, Bathori G, von Richter O, Koomen GJ, Krajcsi P
ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.
Drug Metab Dispos. 2007 Sep;35(9):1533-42. Epub 2007 May 30., [PMID:17537873]

Abstract [show]
The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

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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

[hide] Evidence for dual mode of action of a thiosemicarb... Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3287-96. Wu CP, Shukla S, Calcagno AM, Hall MD, Gottesman MM, Ambudkar SV
Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter.
Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3287-96., [PMID:18089722]

Abstract [show]
Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette (ABC) transporters, including ABCB1 (P-glycoprotein), ABCG2, and several ABCC family members [multidrug resistance-associated protein (MRP)]. We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein, and it resensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4, and MRP5. Our findings showed that NSC73306 is not more toxic to cells that overexpress these transporters compared with their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140-150 nmol/L concentration required for 50% stimulation) and by inhibition of [(125)I]iodoarylazidoprazosin photolabeling (50% inhibition at 250-400 nmol/L) of the substrate-binding site(s). Overall, NSC73306 seems to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4, or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein-overexpressing cells and by acting as a potent modulator that resensitizes ABCG2-expressing cancer cells to chemotherapeutics.

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

[hide] Combined localization and real-time functional stu... Biochem Biophys Res Commun. 2008 Mar 14;367(3):667-73. Epub 2008 Jan 7. Orban TI, Seres L, Ozvegy-Laczka C, Elkind NB, Sarkadi B, Homolya L
Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter.
Biochem Biophys Res Commun. 2008 Mar 14;367(3):667-73. Epub 2008 Jan 7., 2008-03-14 [PMID:18182157]

Abstract [show]
ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.

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

[hide] Homology modeling of breast cancer resistance prot... J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15. Hazai E, Bikadi Z
Homology modeling of breast cancer resistance protein (ABCG2).
J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15., [PMID:18249138]

Abstract [show]
BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.

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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

[hide] ABCG2: structure, function and role in drug respon... Expert Opin Drug Metab Toxicol. 2008 Jan;4(1):1-15. Polgar O, Robey RW, Bates SE
ABCG2: structure, function and role in drug response.
Expert Opin Drug Metab Toxicol. 2008 Jan;4(1):1-15., [PMID:18370855]

Abstract [show]
ABCG2 was discovered in multi-drug-resistant cancer cells, with the identification of chemotherapeutic agents, such as mitoxantrone, flavopiridol, methotrexate and irinotecan as substrates. Later, drugs from other therapeutic groups were also described as substrates, including antibiotics, antivirals, HMG-CoA reductase inhibitors and flavonoids. An expanding list of compounds inhibiting ABCG2 has also been generated. The wide variety of drugs transported by ABCG2 and its normal tissue distribution with highest levels in the placenta, intestine and liver, suggest a role in protection against xenobiotics. ABCG2 also has an important role in the pharmacokinetics of its substrates. Single nucleotide polymorphisms of the gene were shown to alter either plasma concentrations of substrate drugs or levels of resistance against chemotherapeutic agents in cell lines. ABCG2 was also described as the determinant of the side population of stem cells. All these aspects of the transporter warrant further research aimed at understanding ABCG2's structure, function and regulation of expression.

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

[hide] Leflunomide and its metabolite A771726 are high af... Ann Rheum Dis. 2009 Jul;68(7):1201-7. Epub 2008 Apr 8. Kis E, Nagy T, Jani M, Molnar E, Janossy J, Ujhellyi O, Nemet K, Heredi-Szabo K, Krajcsi P
Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.
Ann Rheum Dis. 2009 Jul;68(7):1201-7. Epub 2008 Apr 8., [PMID:18397960]

Abstract [show]
BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.

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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

[hide] Ins and outs of the ABCG2 multidrug transporter: a... Adv Drug Deliv Rev. 2009 Jan 31;61(1):47-56. Epub 2008 Dec 24. Hegedus C, Szakacs G, Homolya L, Orban TI, Telbisz A, Jani M, Sarkadi B
Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):47-56. Epub 2008 Dec 24., 2009-01-31 [PMID:19135105]

Abstract [show]
The major aim of this chapter is to provide a critical overview of the in vitro methods available for studying the function of the ABCG2 multidrug transporter protein. When describing the most applicable assay systems, in each case we present a short overview relevant to ABC multidrug transporters in general, and then we concentrate on the tools applicable to analysis of substrate-drug interactions, the effects of potential activators and inhibitors, and the role of polymorphisms of the ABCG2 transporter. Throughout this chapter we focus on recently developed assay systems, which may provide new possibilities for analyzing the pharmacological aspects of this medically important protein.

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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

[hide] Arginine 383 is a crucial residue in ABCG2 biogene... Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3. Polgar O, Ediriwickrema LS, Robey RW, Sharma A, Hegde RS, Li Y, Xia D, Ward Y, Dean M, Ozvegy-Laczka C, Sarkadi B, Bates SE
Arginine 383 is a crucial residue in ABCG2 biogenesis.
Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3., [PMID:19406100]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

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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

[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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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

[hide] Insect cell versus bacterial overexpressed membran... Methods Mol Biol. 2010;654:47-75. Pozza A, Perez-Victoria JM, Di Pietro A
Insect cell versus bacterial overexpressed membrane proteins: an example, the human ABCG2 transporter.
Methods Mol Biol. 2010;654:47-75., [PMID:20665261]

Abstract [show]
The multidrug resistance phenotype of cancer cells has been often related to overexpression of plasma membrane ATP-binding cassette transporters, which are able to efflux many types of drug by using the energy of ATP hydrolysis. ABCG2 is a half-transporter recently involved. Its purification would help to understand the mechanism of both transport and its inhibition. Biophysical, structural, and functional studies are consuming great amounts of homogeneous purified proteins and require efficient overexpression systems. Heterologous overexpression of human membrane proteins is actually a challenge because these proteins are toxic for the host, and both translation and chaperone systems of the host are not well adapted to the biosynthesis of human proteins. Overexpression of ABCG2 has been assayed in both bacterial and insect cell/baculovirus systems. Although it was highly overexpressed in bacterial system, neither transport nor ATPase activity was found within inverted membrane vesicles. By contrast, insect cells/baculovirus system produces a low amount of protein, a part of which is active.

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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

[hide] Drug delivery across the blood-brain barrier: why ... Expert Opin Drug Deliv. 2006 May;3(3):419-35. Su Y, Sinko PJ
Drug delivery across the blood-brain barrier: why is it difficult? how to measure and improve it?
Expert Opin Drug Deliv. 2006 May;3(3):419-35., [PMID:16640501]

Abstract [show]
The development of drugs that act in the CNS has been significantly impeded by the difficulty of delivering them across the blood-brain barrier (BBB). This article aims to provide the reader with a critical overview of important issues in the discovery and development of drugs that need to enter the brain to elicit pharmacological activity, focusing particularly on i) the role of drug transporters in brain permeation and how to manipulate them to enhance drug brain bioavailability; ii) the successful application, limitations and challenges of commonly used in vitro and in vivo methodologies for measuring drug transport across the BBB, and iii) a discussion of recently developed strategies (e.g., modulation of efflux transporters by chemical inhibitors and the employment of delivery vectors taking advantage of native transport systems at the BBB) for facilitating drug penetration into the brain.

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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

[hide] Functional expression and characterization of the ... Biochem Biophys Res Commun. 2004 Jul 30;320(3):860-7. Cserepes J, Szentpetery Z, Seres L, Ozvegy-Laczka C, Langmann T, Schmitz G, Glavinas H, Klein I, Homolya L, Varadi A, Sarkadi B, Elkind NB
Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization.
Biochem Biophys Res Commun. 2004 Jul 30;320(3):860-7., [PMID:15240127]

Abstract [show]
The closely related human ABC half-transporters, ABCG1 and ABCG4, have been suggested to play an important role in cellular lipid/sterol regulation but no experimental data for their expression or function are available. We expressed ABCG1 and ABCG4 and their catalytic site mutant variants in insect cells, generated specific antibodies, and analyzed their function in isolated membrane preparations. ABCG1 had a high basal ATPase activity, further stimulated by lipophilic cations and significantly inhibited by cyclosporin A, thyroxine or benzamil. ABCG4 had a lower basal ATPase activity which was not modulated by any of the tested compounds. The catalytic site (K-M) mutants had no ATPase activity. Since dimerization is a requirement for half-transporters, we suggest that both ABCG1 and ABCG4 function as homodimers. Importantly, we also found that co-expression of the ABCG4-KM mutant selectively abolished the ATPase activity of the ABCG1 and therefore they most probably also heterodimerize. The heterologous expression, specific recognition, and functional characterization of these transporters should help to delineate their physiological role and mechanism of action.

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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

[hide] ABCG2 is not able to catalyze glutathione efflux a... Front Pharmacol. 2013 Nov 7;4:138. doi: 10.3389/fphar.2013.00138. eCollection 2013. Gauthier C, Ozvegy-Laczka C, Szakacs G, Sarkadi B, Di Pietro A
ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity.
Front Pharmacol. 2013 Nov 7;4:138. doi: 10.3389/fphar.2013.00138. eCollection 2013., [PMID:24312054]

Abstract [show]
ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce "collateral sensitivity" leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and (3)H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.

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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

[hide] Regulation of the function of the human ABCG2 mult... Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2. Telbisz A, Hegedus C, Varadi A, Sarkadi B, Ozvegy-Laczka C
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.
Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2., [PMID:24384916]

Abstract [show]
ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function.

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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

[hide] Determinants of the activity and substrate recogni... Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18. Szafraniec MJ, Szczygiel M, Urbanska K, Fiedor L
Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).
Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18., [PMID:25036722]

Abstract [show]
The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.

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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

[hide] Collateral sensitivity: ABCG2-overexpressing cells... Free Radic Biol Med. 2014 Nov;76:47-52. doi: 10.1016/j.freeradbiomed.2014.07.020. Epub 2014 Jul 23. Krzyzanowski D, Bartosz G, Grzelak A
Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress.
Free Radic Biol Med. 2014 Nov;76:47-52. doi: 10.1016/j.freeradbiomed.2014.07.020. Epub 2014 Jul 23., [PMID:25064323]

Abstract [show]
Multidrug resistance (MDR), which is the main obstacle to cancer chemotherapy, is mainly due to overexpression of ATP-binding cassette (ABC) transporters, especially ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP). A novel idea to overcome MDR is that of collateral sensitivity, i.e., finding a treatment to which cells overexpressing ABC transporters are more sensitive than cells that do not overexpress them. In this study we demonstrate for the first time that MDCKII-BCRP cells, overexpressing ABCG2, are more vulnerable to exogenous oxidative stress induced by several oxidants, viz. paraquat, menadione, hydrogen peroxide, tert-butylperoxide, and 2,2-azobis(2-methylpropionamidine) dihydrochloride. MDCKII-BCRP cells have significantly decreased glutathione level and decreased activities of glutathione S-transferase and glutathione reductase, which may underlie their augmented vulnerability to oxidative stress. These results suggest the possibility of using agents that induce oxidative stress to selectively kill cells overexpressing BCRP.

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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

[hide] Mutations of the central tyrosines of putative cho... Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14. Gal Z, Hegedus C, Szakacs G, Varadi A, Sarkadi B, Ozvegy-Laczka C
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.
Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14., [PMID:25445676]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.

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