Abstract
Purpose
Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients’ responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations.
Methods
ABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.
Results
The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively.
Conclusions
The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.
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Abbreviations
- ABC:
-
ATP-binding cassette
- ABCP:
-
Placenta-specific ABC transporter
- BCRP:
-
Breast cancer resistance protein
- BMA:
-
Bafilomycin A1
- BSA:
-
Bovine serum albumin
- D-MEM:
-
High-glucose Dulbecco’s modified Eagle’s medium
- DTT:
-
Dithiothreitol
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- FCS:
-
Fetal calf serum
- FISH:
-
Fluorescence in situ hybridization
- FRT:
-
Flp recombination target
- HRP:
-
Horseradish peroxidase
- MTT:
-
3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
- MXR:
-
Mitoxantrone resistance-associated protein
- PBS:
-
Phosphate-buffered saline without both Ca2+ and Mg2+
- RT-PCR:
-
Reverse transcriptase-polymerase chain reaction
- SNP:
-
Single nucleotide polymorphism
- TBS:
-
Tris-buffered saline
- TTBS:
-
TBS with 0.05% (v/v) Tween 20
- WT:
-
Wild type
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Acknowledgments
We thank Drs. Kenta Mikuriya, Takayoshi Matsubara, and Satoshi Kometani (Yokogawa Electric Corporation) for generous support in fluorescence-microscopic observations. This study was supported by the NEDO International Joint Research Grant program “International standardization of functional analysis technology for genetic polymorphisms of drug transporters” as well as Grant-in-Aid for Scientific Research (A) (No. 18201041), Grant-in-Aid for Exploratory Research (No. 19659136) from the Japanese Society for the Promotion of Science (JSPS), and Grant-in-Aid for Young Scientists (B) (No. 19791361) from the Ministry of Education, Culture, Sports, Science and Technology. In addition, research in the Osawa laboratory at the University of Michigan Medical School was supported by an NIH grant GM077430.
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Furukawa, T., Wakabayashi, K., Tamura, A. et al. Major SNP (Q141K) Variant of Human ABC Transporter ABCG2 Undergoes Lysosomal and Proteasomal Degradations. Pharm Res 26, 469–479 (2009). https://doi.org/10.1007/s11095-008-9752-7
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DOI: https://doi.org/10.1007/s11095-008-9752-7