Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization.
ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a three-amino acid sequence in transmembrane segment 5 (residues 552-554). Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedly reduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylation was suggested by a reduction in molecular mass compared to that of the 72 kDa wild-type ABCG2. Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein. Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each other but are unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.
Polgar
Orsolya
O
Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 13N240, Bethesda, Maryland 20892, USA.
Ozvegy-Laczka
Csilla
C
Robey
Robert W
RW
Morisaki
Kuniaki
K
Okada
Masaki
M
Tamaki
Akina
A
Koblos
Gabriella
G
Elkind
N Barry
NB
Ward
Yvona
Y
Dean
Michael
M
Sarkadi
Balazs
B
Bates
Susan E
SE
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D019281:Dimerization; D015870:Gene Expression; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D021381:Protein Transport; D016415:Sequence Alignment
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005998:Glycine