Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2.
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.
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.
Ierano
Caterina
C
Tamaki
Akina
A
Stanley
Bradford
B
Ward
Yvona
Y
Xia
Di
D
Tarasova
Nadya
N
Robey
Robert W
RW
Bates
Susan E
SE
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008942:Mitoxantrone / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D013912:Threonine
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D003432:Cross-Linking Reagents; D007976:Leupeptins; D018942:Macrolides; D009363:Neoplasm Proteins; D013912:Threonine; D008942:Mitoxantrone; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde