Arginine 383 is a crucial residue in ABCG2 biogenesis

Abstract

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.

Figure 1

Sequence alignment for members of the ABCG subfamily. Arginine 383 of ABCG2 is well conserved in the ABCG subfamily and in the Drosophila white protein. Arginine 383 and other nearby positively charged residues in ABCG2 are highlighted. The program ClustalX was used to create the alignments.

Figure 2

Surface expression, function, protein and RNA levels of the R383A and R383G mutants transfected into HEK 293 cells. A: Flow cytometry with the 5D3 surface antibody for two clones of each mutant. Stably transfected HEK 293 cells were incubated for 30 min in phycoerythrin-labeled negative control antibody (solid line) or 5D3 antibody (dashed line) and analyzed in a FACSsort flow cytometer. B: Cells were incubated for 30 min in complete media containing 1 μM pheophorbide a or 20 μM mitoxantrone with or without 10 μM of the ABCG2-blocker FTC. (Accumulation without FTC – solid line, with FTC – dashed line.) C: Membrane proteins from the same mutant clones and wild-type were separated by SDS/PAGE (50 μg/lane), transferred onto a PVDF membrane, and probed with the monoclonal anti-ABCG2 antibody BXP-21. D: Immunoblot analysis of membrane proteins from wild-type (50 μg) and R383A (150 μg) transfectants with the BXP-21 following overnight treatment with Endo H, or with N-Glycosisdase F. The second lane for both the wild-type and the mutant represents overnight incubation in buffer with no enzyme. Two independent experiments are shown for the mutant. E: Northern blot showing RNA levels of one clone of each mutant compared to wild-type and empty vector transfected cells (pcDNA). Total RNA (20 μg/lane) from each transfectant was electrophoresed and transferred to a nitrocellulose membrane.

Figure 3

The mechanism of action of kifunensine and MG132 Schematic representation of the pathways blocked by kifunensine and MG132 as part of the Endoplasmic Reticulum Associated Protein Degradation (ERAD).

Figure 4

Proteasome/lysosome inhibition and “rescue” of the R383A mutant with mitoxantrone (MX). Parts A, B, and C: membranes were harvested subsequent to overnight incubation with or without 3μM MG132, or 10 nM bafilomycin (part A), or 30 μg/mL kifunensine (part B), or 5 μM mitoxantrone (part C) followed by immunoblot analysis with the BXP-21 antibody for the wild-type (10 μg/lane) and R383A (10 μg/lane) transfectants. For part C, results of two experiments performed separaretly are shown. The numbers below represent the percentage of detected protein in the upper (N-glycosylated) and lower (non glycosylated) bands as determined by the Odyssey software. Part D: following overnight treatment with 3 μM MG132, or 5 μM mitoxantrone, cells were incubated for 30 min in phycoerythrin-labeled negative control antibody (solid line) or 5D3 antibody (dashed line) and analyzed in a FACSsort flow cytometer for surface expression of ABCG2. (The experiments were performed in triplicates.)

Figure 5

Localization of the R383A mutant in HEK 293 cells. Confocal microscopy of stably transfected HEK 293 was performed following fixation with paraformaldehyde and permeabilization with methanol. Immunostaining was carried out for 1 hour at room temperature with the BXP-21 monoclonal anti-ABCG2 antibody (green) and the anti-calnexin polyclonal antibody (red).

Figure 6

The R383A mutant in Sf9 insect cells. A: Immunodetection of the indicated amounts of membrane proteins with the BXP-21 monoclonal anti-ABCG2 antibody. B: Flow cytometry with the 5D3 monoclonal anti-ABCG2 antibody using non-permeabilized Sf9 cells (as detailed in Figure 2). 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. ATPase activity was measured determining the liberation of inorganic phosphate from ATP with a colorimetric reaction with (grey bars) or without (black bars) the ABCG2-specific inhibitor Ko143.

Figure 7

Surface expression, function and protein levels of the R383K mutant transfected into HEK 293 cells. A: The R383K mutant (clone #8) detected on the cell surface with the 5D3 antibody by flow cytometry performed as described for Figure 2. (Negative control antibody – solid line, 5D3 antibody – dashed line) B: 30 μg of membrane proteins on immunoblot with the BXP-21 antibody. C: Flow cytometry following incubation in complete media containing 250 nM BODIPY-prazosin, or 20 μM mitoxantrone, or 1 μM pheophorbide a, with or without 10 μM of the ABCG2- blocker, FTC. (Accumulation without FTC – solid line, with FTC – dashed line)