Methods to Examine the Impact of Nonsynonymous SNPs on Protein Degradation and Function of Human ABC Transporter

Clinical studies have strongly suggested that genetic polymorphisms and/or mutations of certain ATP-binding cassette (ABC) transporter genes might be regarded as significant factors affecting patients’ responses to medication and/or the risk of diseases. In the case of ABCG2, certain single nucleotide polymorphisms (SNPs) in the encoding gene alter the substrate specificity and/or enhance endoplasmic reticulum-associated degradation (ERAD) of the de novo synthesized ABCG2 protein via the ubiquitin-mediated proteasomal proteolysis pathway. Hitherto accumulated clinical data imply that several nonsynonymous SNPs affect the ABCG2-mediated clearance of drugs or cellular metabolites, although some controversies still exist. Therefore, we recently developed high-speed functional screening and ERAD of ABC transporters so as to evaluate the effect of genetic polymorphisms on their function and protein expression levels in vitro. In this chapter we present in vitro experimental methods to elucidate the impact of nonsynonymous SNPs on protein degradation of ABCG2 as well as on its transport function.