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

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-ABCG2WT 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 A 1 , 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.

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules

“…To investigate the opposing observations and to explore the possibility that both a loss of function and a expression defect could be caused by the Q141K mutation, we expressed our ABCG2 constructs in mammalian cell expression systems. We found, much like previous reports (12,13,15,18), that, in multiple mammalian cell lines, transient expression of the Q141K mutation resulted in significantly less total protein (Fig. S1 A and B and Fig.…”

“…However, in our heterologous Xenopus expression system we did not observe a reduction in protein expression levels in the mutant protein (Fig. 1A), unlike what had been reported previously (12,13,15,18). To investigate the opposing observations and to explore the possibility that both a loss of function and a expression defect could be caused by the Q141K mutation, we expressed our ABCG2 constructs in mammalian cell expression systems.…”

“…Multiple groups reported that the Q141K mutation decreases protein expression and, coincidentally, xenotoxin transport function (11)(12)(13)(14)(15)(16). This decrease of expression and function appears to be partially caused by the enhancement of the Q141K mutant's susceptibility to proteasomal degradation (11)(12)(13).…”

The ABC of glycosylation

“…Indeed, N-linked glycans are thought to be crucial regulators of the stability of ABCG2 in the endoplasmic reticulum 8,9 . In fact, N-glycosylated wildtype ABCG2 is degraded in lysosomes, whereas misfolded mutant proteins have been shown to undergo ubiquitin-mediated degradation in the proteasome 10,11 . We have recently observed that increased levels of glycosylation-defective ABCC1 and ABCC4 are associated with resistance to platinum compounds in ovarian carcinoma cell lines 12 .…”

Multidrug Resistence and Breast Cancer

“…Several studies consistently revealed that Q141K had a lower protein expression level than wild-type BCRP in both transfected cells and human tissues. A recent study has revealed that Q141K undergoes increased lysosomal and proteasomal degradations than wild-type BCRP, possibly explaining the lower level of protein expression of the variant [31]. The R482T and R482G variants of BCRP detected from MCF7/ AdVp3000 and S1-M1-80 cells belong to non-natural mutants.…”