Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity

Abstract

The human breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance and mediates the active efflux of drugs and xenobiotics. BCRP contains one nucleotide-binding domain (NBD) followed by one membrane-spanning domain (MSD). We investigated whether prolines in or near the transmembrane helices are essential for BCRP function. Six proline residues were substituted with alanine individually, and the mutants were stably expressed in Flp-In(TM)-293 cells at levels comparable to that of wild-type BCRP and predominantly localized on the plasma membrane of the cells. While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin. Other mutants had no significant changes in the efflux activities of these substrates. Drug resistance profiles of the cells expressing the mutants correlated well with the efflux data. ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP. These results strongly suggest Pro(392) and Pro(485) are important in determining the overall transport activity and substrate selectivity of BCRP, respectively. Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner. In contrast, mitoxantrone had no significant effect on 5D3 binding. Homology modeling indicates that Pro(392) may play an important role in the communication between the MSD and NBD as it is predicted to be located at the interface between the two functional domains, and Pro(485) induces flexible hinges that may be essential for the broad substrate specificity of BCRP.

Figure 1. Human BCRP topology (A) and sequence alignment (B)

A) The boundary of TM helices is based on the experimentally determined membrane topology of BCRP and only the MSD is shown. Proline residues analyzed in this study are circled and indicated with amino acid positions. B) Human BCRP amino acid sequence in the TM helices was aligned with the corresponding sequences of BCRP homologs and orthologs using Clustal W. Proline residues analyzed in this study are highlighted.

Figure 2. Immunoblotting analysis of the expression levels of wild-type and mutant BCRP

The protein expression levels of wild-type and mutant BCRP in stably transfected Flp-In^™-293 cells were determined by immunoblotting, densitometric analysis of the blots, and normalization to internal standard. A) Immunoblot of whole cell lysates for wild-type and mutant BCRP. Each lane was loaded with 20 μg of protein. B) The protein expression levels of the mutants relative to wild-type BCRP (100%) in whole cell lysates. Shown are means ± SD of three experiments. C) Immunoblot of plasma membrane preparations for wild-type and mutant BCRP. Each lane was loaded with 5 μg of protein. D) The protein expression levels of P392A and P485A relative to wild-type BCRP (100%) in the plasma membrane samples. Shown are means ± SD of three experiments.

Figure 3. Confocal microscopy analysis and cell surface expression of Flp-In^™-293 cells stably expressing wild-type and mutant BCRP

A) The cellular localization of wild-type and mutant BCRP in Flp-In^™-293 cells (shown in green) was determined by immunofluorescent confocal microscopy using the BCRP-specific mAb BXP-21. Cell nuclei were stained with DAPI and are shown in red. B) Expression of wild-type and mutant BCRP on cell surface of stably transfected Flp-In^™-293 cells was detected using the 5D3 monoclonal antibody. Representative flow cytometry histograms showing cell surface expression of wild-type and mutant BCRP are presented. The red and blue peaks represent the phycoerythrin fluorescence associated with cells treated with the IgG2b negative control and the 5D3 antibodies, respectively. No surface expression of BCRP was detected in the pcDNA5 vector control cells.

Figure 4. FTC-inhibitable efflux activities of Flp-In^™-293 cells stably expressing wild-type and mutant BCRP

Three substrates, mitoxantrone (A), BODIPY-prazosin (B), and Hoechst33342 (C) were used. The efflux activities were expressed as the differences in median fluorescence (ΔF) between the FTC/efflux and efflux histograms. Shown are means ± S.D. of three experiments. Differences in efflux activities between wild-type and mutant BCRP are statistically significant: * p < 0.05 by the Student’s t-test. The pcDNA5 vector control, wild-type BCRP, P392A, P480A, P485A, P501A, P574A and P623A are indicated by P, WT, 392, 480, 485, 501, 574 and 623, respectively. Since the expression levels of wild-type and mutant BCRP were comparable, we did not normalize the efflux activity data to BCRP protein expression.

Figure 5. Venadate-sensitive ATPase of plasma membrane preparations containing wild-type and mutant BCRP

ATPase activities were measured using plasma membrane preparations isolated from Flp-In^™-293 cells stably expressing wild-type and mutant BCRP over a range of MgATP concentration (0 – 10 mM). Shown are mean ± S.D. of three experiments. Since the expression levels of wild-type and mutant BCRP were comparable, we did not normalize the ATPase activity data to BCRP protein expression.

Figure 6. Concentration-dependent effects of prazosin and MX on 5D3 binding to wild-type BCRP, P392A, and P485A

The effects of prazosin (A) and MX (B) on 5D3 binding to wild-type and mutant BCRP over a concentration range of 0 – 40 μM and 0 – 80 μM, respectively, were determined using flow cytometry. Shown are means ± S.D. of three experiments. * indicates statistically significant differences between wild-type BCRP and P392A or P485A (p < 0.05 by the Student’s t-test) at various prazosin concentrations.

Figure 7. Schematic illustration of a molecular hinge in TM3 introduced by Pro⁴⁸⁵

A side view of a three-dimensional model of BCRP in the nucleotide-free closed apo inward-facing form is presented. The TM helices are labeled with roman numerals, I, II, III, IV, V and VI. The residues Pro³⁹², Pro⁴⁸⁰ and Pro⁴⁸⁵ are shown in magenta, green and green, respectively, and pointed by arrows. Also shown on the right is an expanded view of TM3 and neighboring helices indicating a potential hinge (shown in green) introduced by Pro⁴⁸⁵. The C-terminal half of TM3 is kinked between TM1 and TM6 in the hinged conformation and rotated closer to the interior of the drug-binding cavity of BCRP. The undistorted TM3 in the original BCRP model is shown in grey.