title: Mutational analysis of ABCG2: role of the GXXXG motif.
abstract: ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.
authors.affiliation: Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Polgar
authors.forename: Robert W
authors.initials: RW
authors.lastname: Robey
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Morisaki
authors.forename: Michael
authors.initials: M
authors.lastname: Dean
authors.forename: Christopher
authors.initials: C
authors.lastname: Michejda
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.forename: Nadya
authors.initials: N
authors.lastname: Tarasova
authors.forename: Susan E
authors.initials: SE
authors.lastname: Bates
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins