ABCMdb

ABCG2 p.Gly406Leu

Predicted by SNAP2:	A: N (72%), C: D (63%), D: D (85%), E: D (91%), F: D (91%), H: D (91%), I: D (85%), K: D (91%), L: D (85%), M: D (80%), N: D (75%), P: D (85%), Q: D (85%), R: D (91%), S: N (53%), T: D (80%), V: D (75%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: D, V: D, W: D, Y: D,

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[hide] Mutational analysis of ABCG2: role of the GXXXG mo... Biochemistry. 2004 Jul 27;43(29):9448-56. Polgar O, Robey RW, Morisaki K, Dean M, Michejda C, Sauna ZE, Ambudkar SV, Tarasova N, Bates SE
Mutational analysis of ABCG2: role of the GXXXG motif.
Biochemistry. 2004 Jul 27;43(29):9448-56., 2004-07-27 [PMID:15260487]

Abstract [show]
ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.

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4 The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Login to comment
5 Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Login to comment
55 The following ABCG2 mutants were generated: G406L, G410L, G406L/G410L, G406A, G410A, and G406A/ G410A. Login to comment
131 This was particularly true for the G406L/G410L double mutants. Login to comment
141 As indicated by almost no change in the level of fluorescence for mitoxantrone in the G406L/G410L mutant, this mutation impairs mitoxantrone transport more than the single mutations. Login to comment
143 As shown in Figure 3 the double leucine mutant does not transport this compound, while the G406L and G410L mutants show impaired transport of pheophorbide a. Login to comment
151 Using DSS, which cross-links proteins 11.4 Å apart, a dimer or higher order multimer was observed by immunoblot analysis in the G406L and G406L/G410L mutants and in the R482G control cells, as shown in Figure 4A. Next, cross-linking studies were performed with DSP, an agent that can be cleaved with the addition of DTT or -mercaptoethanol. Login to comment
155 (C) RNA levels of one representative clone of each of the leucine and alanine mutants on Northern blot with one of the highest levels in the G406L/G410L mutant. Login to comment
161 Transport capacity for BODIPY-prazosin, rhodamine 123, mitoxantrone, and pheohorbide A in the R482G, G406A/G410A, G406L/G410L, G406L, and G410L transfected cells. Plots: Accumulation without FTC (s) and with FTC (---). Login to comment
162 Note lost BODIPY-prazosin and pheophorbide a transport with greatly impaired mitoxantrone transport in the double leucine mutant (G406L/G410L) and almost completely lost rhodamine 123 transport in all leucine mutants. Login to comment
163 The single leucine mutants (G406L, G410L) show impaired transport for BODIPY-prazosin, mitoxantrone, and pheophorbide a when compared to the fully functional R482G control cell line. Login to comment
170 (D) Similar results were obtained for the G406L and G410A mutants. Login to comment
174 Identical results were obtained with all the leucine and alanine mutants (Figure 4C showing results for the G406A and G410L mutants and Figure 4D for G406L and G410A; similar data not shown for the G406L/G410L and G406A/G410A mutants), implying that even if dimerization is impaired in these mutants, it has not prevented their close association on the cell surface. Login to comment
179 We determined the vanadate-sensitive component of the ATPase activity of the G406L/G410L mutant in the presence of 0, 1, 10, and 100 µM prazosin and compared it to the R482G and empty pcDNA3.1 vector transfected cells. Login to comment
181 Significantly, the G406L/G410L mutant, which previously showed the greatest impairment in transport capacity by flow cytometry (Figure 3), revealed ATPase levels identical to the empty vector transfected cell line with no stimulation with prazosin. Login to comment
186 The increased ABCG2 expression after overnight treatment with mitoxantrone in the G406L and G406L/G410L mutants was also confirmed with confocal microscopy (Figure 6B). Login to comment
192 While the double leucine mutant G406L/G410L was found on the cell surface, protein levels were reduced on immunoblot, and transport function and ATP hydrolysis were markedly impaired. Login to comment
201 The vanadate-sensitive ATP hydrolysis in the presence of the indicated concentrations of prazosin for crude membranes of HEK293 cells expressing ABCG2 R482G (0), G406L/G410L mutants (]), and empty vector transfected cells (pcDNA; O) is shown. Login to comment
202 The G406L/G410L mutant demonstrates basal ATPase levels identical to those of the pcDNA with no substrate induction observed. Login to comment
232 (B) Confocal microscopy of HEK293 cells transfected with ABCG2 mutants shows a significant increase in ABCG2 levels for the G406L and G406L/G410L mutants after overnight treatment with 5 µM mitoxantrone. Login to comment
242 Remarkably, both the basal and prazosin-stimulated ATPase activity was found to be severely impaired in the G406L/G410L mutant. Login to comment
247 Since both the basal and the prazosin-stimulated ATP hydrolyses were impaired in the G406L/G410L mutant, it is conceivable that the GXXXG motif could play a similar role in ABCG2. Login to comment

[hide] Functional analysis of the human variants of breas... Drug Metab Dispos. 2005 Jun;33(6):697-705. Epub 2005 Mar 2. Vethanayagam RR, Wang H, Gupta A, Zhang Y, Lewis F, Unadkat JD, Mao Q
Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N.
Drug Metab Dispos. 2005 Jun;33(6):697-705. Epub 2005 Mar 2., [PMID:15743976]

Abstract [show]
Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.

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268 The ATPase activities of the G406L/G410L mutant, which is predicted in the first TM segment of BCRP, were found to be completely abolished (Polgar et al., 2004). Login to comment
269 These data suggest that, whereas ATPase activities of BCRP could be affected by mutations in the NBD (e.g., Q141K and I206L), mutations in other regions including the TM segments (e.g., G406L/G410L) and extracellular loops (e.g., N590Y and D620N) can also influence ATP hydrolysis. Login to comment

[hide] Role of the breast cancer resistance protein (ABCG... AAPS J. 2005 May 11;7(1):E118-33. Mao Q, Unadkat JD
Role of the breast cancer resistance protein (ABCG2) in drug transport.
AAPS J. 2005 May 11;7(1):E118-33., [PMID:16146333]

Abstract [show]
The 72-kDa breast cancer resistance protein (BCRP) is the second member of the subfamily G of the human ATP binding cassette (ABC) transporter superfamily and thus also designated as ABCG2. Unlike P-glycoprotein and MRP1, which are arranged in 2 repeated halves, BCRP is a half-transporter consisting of only 1 nucleotide binding domain followed by 1 membrane-spanning domain. Current experimental evidence suggests that BCRP may function as a homodimer or homotetramer. Overexpression of BCRP is associated with high levels of resistance to a variety of anticancer agents, including anthracyclines, mitoxantrone, and the camptothecins, by enhancing drug efflux. BCRP expression has been detected in a large number of hematological malignancies and solid tumors, indicating that this transporter may play an important role in clinical drug resistance of cancers. In addition to its role to confer resistance against chemotherapeutic agents, BCRP actively transports structurally diverse organic molecules, conjugated or unconjugated, such as estrone-3-sulfate, 17beta-estradiol 17-(beta-D-glucuronide), and methotrexate. BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, in the liver canalicular membrane, and at the luminal surface of the endothelial cells of human brain microvessels. This strategic and substantial tissue localization indicates that BCRP also plays an important role in absorption, distribution, and elimination of drugs that are BCRP substrates. This review summarizes current knowledge of BCRP and its relevance to multidrug resistance and drug disposition.

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No. Sentence Comment
181 The mutants G406L, G410L, and G406L/G410L, particularly the double mutant, lost transport activity for rhodamine 123 and displayed reduced transport for mitoxantrone, pheophorbide a, and BODIPY-prazosine. 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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No. Sentence Comment
175 It is a splicing variant with a somewhat lower expression and lower resistance [23] A347Ta Linker Low drug resistance [42] G406L, G410L, G406L/G410L GXXXG motif TM1 Dimerization in related proteins. 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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No. Sentence Comment
227 406-410) sequence motif in ABCG2 was suggested to form a symmetrical interaction in the TM dimer (Polgar et al., 2004) based on site-directed mutagenesis data (Table 3 and Fig. 6), G406L and G410L substitutions resulted in impaired ATP hydrolysis and substrate transport. Login to comment

[hide] Mutational analysis of threonine 402 adjacent to t... Biochemistry. 2010 Mar 16;49(10):2235-45. Polgar O, Ierano C, Tamaki A, Stanley B, Ward Y, Xia D, Tarasova N, Robey RW, Bates SE
Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2.
Biochemistry. 2010 Mar 16;49(10):2235-45., 2010-03-16 [PMID:20088606]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution, and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We conducted mutational analysis of threonine 402, three residues from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to that of the wild type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization.

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5 On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/ G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. Login to comment
6 The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. Login to comment
7 The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. Login to comment
45 The T402L, T402R, T402L/ G406L/G410L, and T402R/G406L/G410L mutants were generated by site-directed mutagenesis in the pcDNA3.1/Myc-HisA(-) vector (Invitrogen) as previously described (19). Login to comment
47 The R482G control and G406L/G410L mutant were previously generated and characterized (19, 23). Login to comment
92 To study the potential role of this threonine in ABCG2 dimerization, we performed substitutions with a leucine (T402L) or arginine (T402R) substitution or combined these substitutions with the glycine to leucine mutations at the GXXXG motif (T402L/ G406L/G410L and T402R/G406L/G410L) followed by stable transfections in HEK 293 cells. Login to comment
95 We used the previously generated G406L/G410L and R482G transfectants in HEK 293 cells as controls (19). Login to comment
101 The T402L and T402R mutants exhibited levels comparable to the control (R482G), while the double (G406L/G410L) and triple mutants (T402L/G406L/G410L and T402R/G406L/G410L) had significantly decreased levels. Login to comment
103 In the case of T402R/G406L/G410L, the lower band comprised the majority of the detected protein. Login to comment
107 The G406L/G410L and T402L/G406L/G410L mutants displayed substantially lower surface expression levels with the 5D3 antibody. Login to comment
108 In the T402R/G406L/G410L triple mutant, only a slight shift between the negative control(solid line) and the 5D3-labeled histograms (dashed line) was present. Login to comment
117 This was an expected result, given the low levels of expression and the fact that the GXXXG double leucine mutant (G406L/G410L) displayed almost no transport activity (19). Login to comment
125 Minimal change was observed in the molecular mass of ABCG2 in the G406L/G410L mutant after Endo H digestion. Login to comment
130 (A) Cell lysates from stably transfected HEK 293 cells (20 μg for R482G, T402L, and T402R and 60 μg for G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L) were separated via SDS-PAGE, transferred onto a PVDF membrane, and probed with monoclonal anti-ABCG2 antibody BXP-21. Login to comment
136 In contrast, the protein levels of the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/ G410L mutants were little affected by bafilomycin treatment (Figure 3). Login to comment
141 Previously, we reported that the levels of the G406L/G410L mutant significantly increased on immunoblot and on immunofluorescence following MX treatment (19). Login to comment
143 As previously shown, a significant increase in protein levels was observed for the G406L/ G410L mutant (Figure 4A). Login to comment
144 In contrast, overnight treatment with MX resulted in a majority of the T402L/G406L/G410L mutant being detected in the normal 72 kDa band as opposed to the double band observed without drug exposure, representing a shift from the lower to the upper molecular mass band (Figure 4A). Login to comment
145 The average percent of mutant protein detected in the lower and upper bands with and without MX is presented in Figure 4B. Notably, the results obtained with the arginine triple mutant (T402R/G406L/G410L) revealed little shift of the lower band, consistent with a more profound defect. Login to comment
148 The G406L/G410L mutant exhibited primarily plasma membrane staining with little intracellular signal and after MX treatment displayed a slightly increased level of surfaceexpression.InthecaseoftheT402L/G406L/G410Lmutant, both intracellular and cell surface staining could be observed prior to treatment with MX, while after overnight MX treatment, we observed an increased level of ABCG2 plasma membrane localization in the T402L/G406L/G410L mutant (Figure 5). Login to comment
149 In contrast, after treatment with MX, the T402R/G406L/G410L mutant still primarily displayed intracellular localization. Login to comment
151 As demonstrated in Figure 6, following MX treatment, the T402L/G406L/G410L mutant was no longer sensitive to Endo H (Figure 6B), implying that its glycan has matured and that the protein has most likely moved out of the ER. Login to comment
152 In contrast, the lower-molecular mass form of the T402R/G406L/G410L mutant, which did not shift to the upper 72 kDa band in the presence of MX, was still sensitive to digestion with Endo H (Figure 6B). Login to comment
155 Immunoblot analysis of cell lysates from the R482G, T402L, and T402R mutants [(A) 35 μg] and from the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants [(B) 70 μg] with the BXP-21 antibody following overnight treatment with Endo H or N-glycosidase F. Login to comment
158 Cells were harvested after overnight incubation with or without 10 nM bafilomycin followed by immunoblot analysis with the BXP-21 and GAPDH antibodies for the R482G, T402L, and T402R mutants (40 μg/lane) and the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants (100 μg/lane). Login to comment
170 Figure 7 demonstrates increased molecular mass bands consistent with cross-linking of the control, T402R, and T402R/ G406L/G410L proteins. Login to comment
171 Similar results were obtained with the T402L and T402L/G406L/G410L mutants with DSG; furthermore, the triple mutants were also cross-linked by DSS at both 4 °C and room temperature (data not shown). Login to comment
180 In contrast, G406L/G410L and T402R/G406L/G410L demonstrated an approximately 50% or more decrease in their CAT activity, implying impaired dimerization of these mutant TMs. Login to comment
226 (B) CAT activity measured in the TOXCAT assay for the G406L/G410L, T402R, and T402R/G406L/G410L mutants compared to that of wild-type TM1 of ABCG2. Login to comment
228 This point is supported by the efficient shift of the lower band in the MX rescue experiments for the T402L and T402L/G406L/ G410L mutants discussed below. Login to comment
233 Our results with the ABCG2 triple mutants carrying the threonine 402 to leucine or arginine substitutions (T402R/ G406L/G410L and T402R/G406L/G410L) are consistent with destabilization of the homodimer. Login to comment
246 (F) Close-up view of the T402L, G406L, and G410L mutations. Login to comment
256 As noted above, the T402L/G406L/G410L mutant could be rescued by overnight treatment with the substrate MX as suggested by its shift to the normal 72 kDa molecular mass band on immunoblot and its loss of Endo H sensitivity. Login to comment
258 In contrast, the T402R/G406L/G410L mutant, carrying the more drastic substitution at residue 402, did not show the same phenomenon (Figures 4-6). Login to comment

[hide] Dimerization of ABCG2 analysed by bimolecular fluo... PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3. Haider AJ, Briggs D, Self TJ, Chilvers HL, Holliday ND, Kerr ID
Dimerization of ABCG2 analysed by bimolecular fluorescence complementation.
PLoS One. 2011;6(10):e25818. Epub 2011 Oct 3., [PMID:21991363]

Abstract [show]
ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.

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164 However, it remains possible that the effects of multiple mutations to a predicted interfacial site could be detected by BiFC, e.g. the surface of helix TM1 which contains a T402L/G406L/G410L dimerization motif [17]. Login to comment

[hide] Genetics of hyperuricemia and gout: implications f... Curr Rheumatol Rep. 2013 Feb;15(2):309. doi: 10.1007/s11926-012-0309-8. George RL, Keenan RT
Genetics of hyperuricemia and gout: implications for the present and future.
Curr Rheumatol Rep. 2013 Feb;15(2):309. doi: 10.1007/s11926-012-0309-8., [PMID:23307580]

Abstract [show]
Gout is the most common inflammatory arthropathy and occurs in the setting of elevated serum urate levels. Gout is also known to be associated with multiple comorbidities including cardiovascular disease and the metabolic syndrome. Recent advances in research have increased our understanding and improved our knowledge of the pathophysiology of gout. Genome-wide association studies have permitted the identification of several new and common genetic factors that contribute to hyperuricemia and gout. Most of these are involved with the renal urate transport system (the uric acid transportasome), generally considered the most influential regulator of serum urate homeostasis. Thus far, SCL22A12, SCL2A9, and GLUT9 have been found to have the greatest variation and most influence on serum urate levels. However, genetics are only a part of the explanation in the development of hyperuricemia and gout. As results have been mixed, the role of known urate influential genes in gout's associated comorbidities remains unclear. Regardless, GWAS findings have expanded our understanding of the pathophysiology of hyperuricemia and gout, and will likely play a role in the development of future therapies and treatment of this ancient disease.

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198 In the context of gout, the ABCG2 ligand mitoxantrone (an ABCG2 ligand) improved the altered protein structure of the T402L-G404L-G406L-G410L mutant in vitro [66]. Login to comment

[hide] Molecular pharmacology of ABCG2 and its role in ch... Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10. Stacy AE, Jansson PJ, Richardson DR
Molecular pharmacology of ABCG2 and its role in chemoresistance.
Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10., [PMID:24021215]

Abstract [show]
The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) is an important member of the ABC transporter superfamily, which has been suggested to be involved in multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many common chemotherapeutics such as imatinib, doxorubicin, and mitoxantrone. Physiologically, ABCG2 is highly expressed in areas such as the blood-brain barrier and gastrointestinal tract, where it is thought to play a role in protection against xenobiotic exposure. High ABCG2 expression has also been found in a variety of solid tumors and in hematologic malignancies and has been correlated with poorer clinical outcomes. Furthermore, ABCG2 expression is a characteristic feature of cancer stem cells, which are able to self-renew and differentiate. These cancer stem cells have been postulated to play an important role in MDR, where their inherent ABCG2 expression may allow them to survive chemotherapy and repopulate the tumor after exposure to chemotherapeutics. This observation raises the exciting possibility that by inhibiting ABCG2, cancer stem cells and other cancers may be targeted and eradicated, at which point conventional chemotherapeutics would be sufficient to eliminate the remaining tumor cells. Inhibitors of ABCG2, such as tyrosine kinase inhibitors, phosphodiesterase-5 inhibitors, and the fumitremorgin-type indolyl diketopiperazine, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxo pyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], could potentially be used for this purpose. However, these agents are still awaiting comprehensive clinical assessment.

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73 Mutational analysis of Thr402, which is located near the GXXXG motif (TXXGXXXG), in combination with mutations of the GXXXG motif (T402L or T402R and G406L or G410L; Fig. 3) resulted in a reduction in protein expression and drug efflux, alterations in glycosylation, and retention of ABCG2 in the ER (Polgar et al., 2010). 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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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
221 The substitution of a single or both Gly residues by Leu in this motif resulted in a loss of transport for rhodamine 123, and impaired transport of mitoxantrone, Pheide and BODIPY-prazosin, in particular, in the double mutant (Gly406 Leu/ Gly410 Leu). Login to comment