Abstract
The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.
Highlights
Multidrug resistance (MDR) represents a serious problem in cancer chemotherapy
The ABCG2-K86M mutant was tagged at the N-terminus with the hemagglutinin (HA) epitope whereas ABCG2-wt was tagged with a MYC epitope
All constructs including untagged ABCG2-wt were expressed in HEK293 cells and the resulting cell lines were analyzed by SDS-PAGE and western blotting of membranes using a polyclonal antibody Ab-405 (Litman et al, 2002) directed against an intracellular epitope of ABCG2
Summary
The resistant tumor cells often overexpress one of several ATP binding cassette (ABC) transporters that are capable of mediating efflux of many clinically important drugs (Litman et al, 2001). These transporters include among others P-glycoprotein, the multidrug resistance associated protein 1 (MRP1) and ABCG2. It should be noted that an amino acid substitution at position 482 distinguishes MXR (R482G), BCRP (R482T) and ABCP (R482, wt) (Allikmets et al, 1998; Doyle et al, 1998; Miyake et al, 1999), which are synonymous designations for ABCG2 This substitution has been shown to influence the substrate specificity of the transporter (Chen et al, 2003b; Mitomo et al, 2003; Ozvegy et al, 2002; Robey et al, 2003)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
