Assessing the ABCG2 Protein Interactome: a Potential Strategy to Disrupt ABCG2 Function

Abstract

The ATP‐binding cassette sub‐family G member 2, ABCG2, is an important plasma membrane transporter that removes potentially harmful endogenous toxins and xenobiotics from cells. ABCG2 was identified as an important contributor to the multidrug resistance phenotype in cancer cells because it exported a wide variety of structurally diverse chemotherapeutic agents, including mitoxantrone, topotecan and the tyrosine kinase inhibitors. High ABCG2 expression in a variety of tumours is associated with poor clinical outcomes. In our present study, we hypothesized that proteins that interact with ABCG2 modulate its function. This knowledge would allow us to discover novel therapeutic targets that could be exploited to alter ABCG2 expression and/or function.To identify interacting proteins for ABCG2, we employed a site‐specific biotinylation system utilizing a 15 amino acid peptide tag known as AviTag, fused to ABCG2. In the presence of the E. coli biotin ligase (BirA), biotin can be covalently attached to the AviTag peptide and the complex can be purified using the non‐covalent streptavidin‐biotin interaction. A plasmid harboring both AviTag‐ABCG2 and an IRES directed BirA was constructed. We stably expressed it in HEK293 cells and confirmed that AviTag ABCG2 was expressed at the plasma membrane and retained typical ABCG2 transport activity. Importantly, the cells expressing the AviTag‐ABCG2 and BirA readily labelled AviTag‐ABCG2 with biotin. The AviTag‐ABCG2 protein complexes captured by streptavidin agarose beads were purified and eluted according to an empirically developed protocol.Using a proteomic approach with mass spectrometry, we identified 39 proteins (both known and novel ABCG2 interacting proteins) at a p‐value threshold of 0.05 in the AviTag‐ABCG2 protein complex compared to the negative control. As expected, ABCG2 was one of the top 10 proteins in abundance (high spectral counts). Among the 39 interacting proteins, we confirmed by immunoblotting that several important cancer‐related proteins interact with ABCG2. These proteins appear capable of regulating ABCG2 function in cancer cells.Future studies will elucidate how these interactions affect ABCG2 function. These findings might aid in the development of new strategies for blocking ABCG2 function in multidrug resistant cancers, which could have important implications in improving patient’s response to chemotherapy.Support or Funding InformationThis work was supported by NIH and ALSAC