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Major SNP (Q141K) Variant of Human ABC Transporter ABCG2 Undergoes Lysosomal and Proteasomal Degradations

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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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