ABCMdb

ABCG2 p.Arg482Ala

Predicted by SNAP2:	A: D (91%), C: D (85%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (85%), K: D (85%), L: D (91%), M: D (85%), N: D (95%), P: D (95%), Q: D (95%), S: D (91%), T: D (91%), V: D (91%), 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, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Single amino acid substitutions in the transmembra... Int J Cancer. 2003 Dec 10;107(5):757-63. Miwa M, Tsukahara S, Ishikawa E, Asada S, Imai Y, Sugimoto Y
Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.
Int J Cancer. 2003 Dec 10;107(5):757-63., 2003-12-10 [PMID:14566825]

Abstract [show]
Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
42 Because FACS analysis showed that certain populations of the methotrexate-resistant PA/R482A and PA/R482W cells did not express BCRP (data not shown), all the R482-mutant BCRP transfectants (except R482K-BCRP transfectant) were further selected with 1 ng/ml of mitoxantrone for 5 days to eliminate untransfected cells. Login to comment
64 PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D (Group 2) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment
163 Group 2 members (PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/ R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment

[hide] Flow cytometry-based approach to ABCG2 function su... Cytometry A. 2004 Dec;62(2):129-38. Garcia-Escarp M, Martinez-Munoz V, Sales-Pardo I, Barquinero J, Domingo JC, Marin P, Petriz J
Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.
Cytometry A. 2004 Dec;62(2):129-38., [PMID:15517563]

Abstract [show]
BACKGROUND: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. METHODS: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter. RESULTS: ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays. CONCLUSIONS: Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
117 KB cells transfected with ABCG2-R482A, R482G, and R482T were seeded in the presence of 0.8 ␮M MXR for 1 h at 37°C. Login to comment
154 Using ABCG2 variant-transfected KB cells, we clearly demonstrated that MXR retention is significantly lower than that observed with wt R482A (MXRA) cells. Login to comment

[hide] Substrate Affinity of Photosensitizers Derived fro... Mol Pharm. 2010 Sep 1. Morgan J, Jackson JD, Zheng X, Pandey SK, Pandey RK
Substrate Affinity of Photosensitizers Derived from Chlorophyll-a: The ABCG2 Transporter Affects the Phototoxic Response of Side Population Stem Cell-like Cancer Cells to Photodynamic Therapy.
Mol Pharm. 2010 Sep 1., 2010-09-01 [PMID:20684544]

Abstract [show]
Photosensitizers (PS) synthesized with the aim of optimizing photodynamic therapy (PDT) of tumors do not always fulfill their potential when tested in vitro and in vivo in different tumor models. The ATP-dependent transporter ABCG2, a multidrug resistant pump expressed at variable levels in cancerous cells, can bind and efflux a wide range of structurally different classes of compounds including several PS used preclinically and clinically such as porphyrins and chlorins. ABCG2 may lower intracellular levels of substrate PS below the threshold for cell death in tumors treated by PDT, leaving resistant cells to repopulate the tumor. To determine some of the structural factors that affect substrate affinity of PS for ABCG2, we used an ABCG2-expressing cell line (HEK 293 482R) and its nonexpressing counterpart, and tyrosine kinase ABCG2 inhibitors in a simple flow cytometric assay to identify PS effluxed by the ABCG2 pump. We tested a series of conjugates of substrate PS with different groups attached at different positions on the tetrapyrrole macrocycle to examine whether a change in affinity for the pump occurred and whether such changes depended on the position or the structure/type of the attached group. PS without substitutions including pyropheophorbides and purpurinimides were generally substrates for ABCG2, but carbohydrate groups conjugated at positions 8, 12, 13, and 17 but not at position 3 abrogated ABCG2 affinity regardless of structure or linking moiety. At position 3, affinity was retained with the addition of iodobenzene, alkyl chains and monosaccharides, but not with disaccharides. This suggests that structural characteristics at position 3 may offer important contributions to requirements for binding to ABCG2. We examined several tumor cell lines for ABCG2 activity, and found that although some cell lines had negligible ABCG2 activity in bulk, they contained a small ABCG2-expressing side population (SP) thought to contain cells which are responsible for initiating tumor regrowth. We examined the relevance of the SP to PDT resistance with ABCG2 substrates in vitro and in vivo in the murine mammary tumor 4T1. We show for the first time in vivo that the substrate PS HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) but not the nonsubstrate PS HPPH-Gal (a galactose conjugate of HPPH) selectively preserved the SP which was primarily responsible for regrowth in vitro. The SP could be targeted by addition of imatinib mesylate, a tyrosine kinase inhibitor which inhibits the ATPase activity of ABCG2, and prevents efflux of substrates. A PDT resistant SP may be responsible for recurrences observed both preclinically and clinically. To prevent ABCG2 mediated resistance, choosing nonsubstrate PS or administering an ABCG2 inhibitor alongside a substrate PS might be advantageous when treating ABCG2-expressing tumors with PDT.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
343 A: HEK-293 R482A. Login to comment

[hide] ABCG2 is required to control the sonic hedgehog pa... Cytometry A. 2011 Sep;79(9):672-83. doi: 10.1002/cyto.a.21103. Epub 2011 Jul 19. Balbuena J, Pachon G, Lopez-Torrents G, Aran JM, Castresana JS, Petriz J
ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.
Cytometry A. 2011 Sep;79(9):672-83. doi: 10.1002/cyto.a.21103. Epub 2011 Jul 19., [PMID:21774076]

Abstract [show]
The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
109 KB cells transfected with ABCG2- R482A were incubated with 1 lg ml21 Ho342 for 3 h at 378C in the presence or absence of 10 or 30 lM cyclopamine. Login to comment