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First published online 15 March 2005
doi: 10.1242/jcs.01729

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Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

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

View larger version (23K): [in a new window]   Fig. 1. The K86M mutation in ABCG2 results in a non-functional transporter. (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. The membrane fractions were analyzed by western blotting using the polyclonal antibody Ab-405 (Litman et al., 2002). (B) ATPase activity assay on isolated membrane fractions. The ATPase activity was measured as release of inorganic phosphate using a colorimetric assay. 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. The experiment shown is representative of at least three other independent experiments. (C) Cytotoxicity assay was used to detect resistance of the stably transfected HEK293 cells to mitoxantrone. Stably transfected cells were incubated for 3 days with mitoxantrone and fixed in 50% TCA. 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. The experiment shown (mean±s.e.m., n=3) is representative of four independent experiments.

View larger version (35K): [in a new window]   Fig. 2. HEK293 cells transfected with ABCG2-K86M display no transport activity. The cells were incubated for 30 minutes at 37°C (accumulation) with 100 nM BODIPY-prazosin in the presence or absence of the specific ABCG2 inhibitor Fumitremorgin C (FTC) (5 µM). The cells were washed and incubated with or without 5 µM FTC for 60 minutes at 37°C (efflux). The histograms show intracellular fluorescence after accumulation and efflux of BODIPY-prazosin (100 nM) in the presence (broken line) or absence (unbroken line) of 5 µM FTC. Fluorescence was measured (FL-1) after excitation with a 488 nm argon laser.

View larger version (39K): [in a new window]   Fig. 3. ABCG2-wt and ABCG2-K86M co-immunoprecipitate. Total cell lysates from stably transfected HEK293 cells were immunoprecipitated (IP) with anti-MYC antibody and protein G agarose. All cell lysates contained 5 mM N-Ethylmaleimide to inhibit spontaneous cysteine oxidation. The eluate was analyzed by immunoblotting (IB) with either anti-HA or anti-MYC (cmyc) antibodies. Cell lysates were loaded on SDS-PAGE as positive controls for expression of both constructs (lane 7, 8). Singly transfected cells were used as negative controls (lane 3, 4).

View larger version (49K): [in a new window]   Fig. 4. ABCG2 exists as a disulfide-linked dimer. Total cell lysates from stably transfected HEK293 cells were analyzed by SDS-PAGE and western blotting. To avoid formation of non-natural disulfide bridges 10 mM N-ethylmaleimide was included in both the lysis buffer and in the SDS loading buffer. The samples were incubated in the SDS loading buffer with or without 100 mM DTT as indicated for 30 minutes before application to the gel. For the immunoblotting we used both HA antibody (upper panel, IB:HA) and MYC antibody (lower panel, IB:cmyc).

View larger version (28K): [in a new window]   Fig. 5. Two functional NBDs are necessary for proper ATP hydrolysis. (A) Membrane fractions from stably co-transfected HEK293 cells. The blots are probed with BXP-21 (total ABCG2), MYC (cmyc) or HA antibodies. In all blots an anti-ß-actin antibody was included as a loading control. (B) Relative expression of HA-tagged and MYC-tagged ABCG2 in co-transfected cells. The expression was assessed by densitometry analysis of western blots of the co-transfected cells blotted with either an anti-MYC or an anti-HA antibody. The histograms are mean±s.e.m. expression normalized to ABCG2-wt-MYC (n=3). (C) ATPase activity measurements on membrane fractions analyzed. Phosphate release was measured in response to increasing concentrations of prazosin. Data are mean±s.e.m. of four experiments performed in triplicate. The EC50 (µM) and Vmax (nmol phosphate/minute/mg protein) were determined by non-linear regression analysis using Sigmaplot.

View larger version (71K): [in a new window]   Fig. 6. ABCG2-K86M is localized in an intracellular compartment. HEK293 cells stably expressing different ABCG2 constructs were analyzed by immunocytochemistry to detect localization of the transporter. The cells were fixed in formaldehyde and stained with anti-ABCG2 monoclonal antibody and a secondary Alexa 488 antibody. The samples were visualized by confocal microscopy. (A) ABCG2-wt, (B) ABCG2-wt-HA, (C) ABCG2-wt-MYC (ABCG2-wt-cmyc), (D) ABCG2-K86M and (E) ABCG2-K86M-HA.

View larger version (31K): [in a new window]   Fig. 7. ABCG2-K86M shows a markedly reduced surface expression. (A) Surface expression of ABCG2 in the stably transfected HEK293 cells were determined by flow cytometry. The cells were stained with PE-conjugated anti-ABCG2 antibody directed against the extracellular domain of ABCG2. Dead cells were detected and omitted by staining with Propidium Iodide. An IgG isotype control antibody was used to detect unspecific binding. The histogram represents the surface expression (mean fluorescence) normalized to ABCG2-wt-MYC (ABCG2-wt-cmyc) (n=3). (B) HEK293 cells stably expressing ABCG2-K86M or ABCG2-wt-MYC + ABCG2-K86M-HA were analyzed by immunocytochemistry to detect localization of the transporter. The cells were stained with anti-ABCG2 or anti-HA antibody and a secondary Alexa 488 antibody. The samples were analyzed using an Axiovert 200 epifluorescence microscope.

View larger version (41K): [in a new window]   Fig. 8. The ABCG2-K86M mutant resides in the ER. Immunocytochemistry co-stainings were performed using an Anti-ABCG2 antibody and antibodies directed against different cellular organelles. Left-hand column, ABCG2 staining; middle column, organelle staining; and right-hand column, overlay of the single stainings. (A,B) Anti-Calnexin (ER), (C,D) Giantin (cis-Golgi) and (E,F) Rab5 (early endosomes). Secondary antibodies were Alexa 488 and Alexa 568. The pictures were obtained by confocal laser-scanning microscopy on a Zeiss LSM 510 confocal microscope.

View larger version (21K): [in a new window]   Fig. 9. ABCG2-K86M is glycosylated to the same degree as ABCG2-wt. (A) Deglycosylation of ABCG2 protein with PNGase F or endonuclease H (Endo H). Total cell lysates of ABCG2-wt or ABCG2-K86M were treated with denaturation buffer for 10 minutes at 100°C. 15 µg total protein were incubated for 2 hours at 37°C with 100 mU enzyme. Samples were analyzed by SDS-PAGE and western blotting using the monoclonal anti-ABCG2 antibody BXP-21. (B) Positive control for endonuclease H activity. Immature IL2R (at 35-40 kDa) is sensitive to endonuclease H and is fully deglycosylated whereas the mature IL2R (50-60 kDa) is insensitive to endonuclease H treatment.

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