Abstract 874: Mutations of aromatic amino residue to polar residue in NBDs alter the transport properties of ABCC10.

Abstract

Abstract ABCC10 is an efflux pump that confers multidrug resistance to cells by extruding a variety of natural and nucleosides analogues using energy from ATP hydrolysis. The objective of this project is to understand the detailed relationship between ATP hydrolysis and drug transport for ABCC10 and how ABCC10’s ATPase activity is regulated. For this study, we mutated aromatic residues to polar residues in the Nucleotide Binding Domains (NBDs) of ABCC10 to determine how these residues are involved in the transport of ABCC10 substrates. Prior, structural analyses of several bacterial ABC-transporters indicated that aromatic amino acid residues in NBDs are needed for ATP binding and ATP hydrolysis. In these studies, substitution of these aromatic residues completely abolished ATP-dependent transport. Prior work on ABCC1 has shown that substitutions such as W (tryptophan) to C (cytidine) or Y (tyrosine) to C (cytidine) decreased ABCC1’s affinity for ATP, and ATP-dependent LTC4 transport activities. Analogous substitutions of W (tryptophan) residues to polar residues in the NBD2 of ABCC2 eliminated estradiol glucuronide and leukotriene C4 transport. Based on these data, we hypothesize that mutating aromatic to polar residues in the NBDs of ABCC10 will modulate the biochemical and cellular transport activities of this efflux pump. At present we have generated mutant-ABCC10 constructs by site-directed mutagenesis. Specifically, we mutated the aromatic Tyr747 (NBD1), Trp607 (NBD1) and Tyr1255 (NBD2) residues to polar cysteine residues. We then examined all ABCC10-mutant, wild-type ABCC10 and empty-vector constructs using immunofluorescence, ATPase assays, sensitivity and transport assays. We observed that the mutant ABCC10 proteins localized predominantly to the membranes. Further, ABCC10-mutant cells, except for the W607C mutant clone, showed less sensitivity than empty vector-transfected cells. Using accumulation assays we also demonstrated that substitutions of aromatic to polar residues, such as W607C and Y1255C, decreased the ability of ABCC10 to transport, docetaxel and Ara-C, however transport was not completely abolished. Using our ABCC10 mutants we found that aromatic to polar residue substitutions eliminated the stimulatory effects of docetaxel and Ara-C on ATPase activity of ABCC10. Taken together, these results demonstrate that these aromatic residues are involved in the ATP binding process and mutations of these residues cause of reduction in ABCC10 transport of docetaxel and Ara-C from cells. Citation Format: Ekaterina V. Malofeeva, Elizabeth Hopper-Borge. Mutations of aromatic amino residue to polar residue in NBDs alter the transport properties of ABCC10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 874. doi:10.1158/1538-7445.AM2013-874