Characterization of Polarized Expression of Point- or Deletion-Mutated Human BCRP/ABCG2 in LLC-PK1 Cells

Abstract

In polarized cells, such as hepatocytes and intestinal epithelial cells, transporters are localized on the apical or basolateral membranes and play important roles in the vectorial transport of their substrates. In the current study, we have aimed to clarify the mechanism for the cellular sorting of human breast cancer resistance protein (BCRP/ABCG2), which is expressed on the apical membrane of many tissues and functions as an efflux transporter. After the expression vector, including wild type or mutants of human BCRP cDNA, was transfected into LLC-PK1 cells, immunohistochemical staining and Western blot analyses were performed to characterize the cellular localization and the status of BCRP, respectively. The transfected cDNA product of wild-type BCRP was expressed on the apical membrane in LLC-PK1 cells. Glycosylation consensus sequences-disrupted mutants showed the apical localization as the wild type, whereas the apical-selective expression disappeared when disulfide bonds could not be formed. Furthermore, examination of the localization of deletion mutants of human BCRP emphasized the importance of some peptide sequences. The region between the N-terminal and ATP-binding cassette and proximal C-terminal region, both of which are well conserved in various animal species, were found to be significant for proper localization. These results suggest that, although the presence of N-glycan does not affect the localization of BCRP, disulfide bonds and some peptide sequences in both the N- and C-terminals are necessary for the apical expression of BCRP.