Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2

Abstract

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.

Figure 1. Protein levels, surface expression, and function of the mutants

Part A. Cell lysates from stably transfected HEK 293 cells (20 μg for R482G, T402L, T402R and 60μg for G406L/G410L, T402L/G406L/G410L, T402R/G406L/G410L mutants) were separated by SDS/PAGE, transferred onto a PVDF membrane, and probed with the monoclonal anti-ABCG2 antibody BXP-21. Part B. First column: flow cytometry with the 5D3 surface antibody. 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 on a FACSsort flow cytometer. Second, third and fourth colums: cells were incubated for 30 min in complete media containing 20 μM MX, or 1 μM pheophorbide a, or 200 nM BODIPY-prazosin with or without 10 μM of the ABCG2-blocker FTC. (Accumulation without FTC - solid line, with FTC - dashed line).

Figure 2. Enzymatic deglycosylation with N-glycosidase F and Endo H

Immunoblot analysis of cell lysates from the R482G, T402L and T402R mutants (panel A, 35 μg) and from the G406L/G410L, T402L/G406L/G410L and T402R/G406L/G410L mutants (panel B, 70 μg) with the BXP-21 antibody following overnight treatment with Endo H, or with N-Glycosidase F. The arrows point out the three different molecular weights observed in the experiment.

Figure 3. Effect of lysosome inhibition on the protein levels of ABCG2 mutants

Cells were harvested subsequent to 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 for the G406L/G410L, T402L/G406L/G410L, T402R/G406L/G410L mutants (100 μg/lane).

Figure 4. “Rescue” of the triple mutants with MX

Part A. Following overnight incubation with or without 5 μM MX, cells were harvested and the lysates (25 μg for R482G, T402R, T402L and 50 μg for the other mutants) were subjected to immunoblot analysis with the BXP-21 and GAPDH antibodies as described in Figure 1. Part B. Average percent of mutant protein detected in the lower and upper bands with and without MX for at least seven separate experiments.

Figure 5. Localization of the mutants in HEK 293 cells after treatment with MX

Confocal microscopy of stably transfected HEK 293 after overnight treatment with or without 5 μM MX 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 (red).

Figure 6. Endo H digestion of the triple mutants following MX treatment

After overnight incubation with or without 5 μM MX, cells were harvested and the lysates were subjected to overnight digestion with Endo H followed by immunoblot analysis with the BXP-21 antibody (panel A, 35 μg for R482G, T402R and T402L and panel B, 70 μg for the double and triple mutants).

Figure 7. Cross-linking studies

Cross-linking was performed on intact cells at room temperature for 30 min with 2 mM DSG followed by protein extraction, SDS/PAGE separation and immunoblotting with the BXP-21 anti-ABCG2 antibody. Cross-linking is observed in both the control, T402L, T402R, and the triple mutants as suggested by higher molecular weight species. The molecular weight of monomeric wild-type ABCG2 is 72 kDa.

Figure 8. The TOXCAT assay

Part A. Schematic representation of the assay. TM alpha helices are fused with the ToxR domain on one end and with the maltose binding protein on the other. If dimerization of the TMs occurs, ToxR forms an active dimer and CAT is transcribed. Part B. The CAT activity measured in the TOXCAT assay for the G406L/G410L, T402R, and T402R/G406L/G410L mutants compared to wild-type TM1 of ABCG2.

Figure 9. Homology modeling of ABCG2

Part A. A cross section of the ABCG2 homology model is given showing the TXXXGXXXG motif and its vicinity including TM1 and 2 from one subunit (Dark red) and TM3, 5 and 6 from another (Dark blue). A putative drug-binding cavity below the residue T402 is also illustrated. Part B. View of TMD of ABCG2 model along the direction perpendicular to the membrane plane shown the arrangement of 12 TM helices and the position of the TXXXGXXXG motif. Part C. A close-up view of the wild-type TXXXGXXXG motif. Part D. A close-up view of the T402L mutation. Part E. A close-up view of the T402R mutation. Part F. A close-up view of the T402L/G406L/G410L mutations.