Our long-term objective is to understand how bile acids modify cholangiocyte biology. We continue to focus on bile acid interactions with cholangiocytes, the epithelial cells lining intrahepatic bile ducts, because of the development of new hypotheses and techniques regarding the regulation of bile acid transport by posttranslational modification of the apical bile acid transporter (ASBT). We have found ASBT protein expression in the cholangiocyte apical membrane is rapidly regulated by a reversible process (due to apical membrane recycling) and by a nonreversible process (due to ubiquitination). This rapid regulation of ASBT is important to changes in bile secretion and to the adaptive response to .cholestasis and inflammation. In the first specific aim, the hypothesis that ASBT recycles between intracellular and apical membranes will be tested by novel technology using apical membrane biotinylation and fluorescence microscopy and polarized cells expressing ASBT-GFP. In the second specific aim, the hypothesis that ASBT phosphorylation and monoubiquitination regulates ASBT endocytosis/exocytosis will be tested by examining phosphorylation, monoubiquitination and ASBT translocation in polarized cells expressing wild, phosphorylation deficient and constitutively phosphorylated mutants or mutants that lack lysine ubiquitination sites. In the third specific aim, the hypothesis that phosphorylation triggers ASBT ubiquitination and proteasome degradation will be tested by determining ASBT polyubiquitination in normal cells or ubiquitination-deficient cells, by strategic lysine replacements mutations to test if there is a 'lysine destruction motif adjacent to a regulatory phosphorylation site, and studies to determine the intracellular site, where ASBT is ubiquitinated: The results of these studies will clarify the mechanisms for posttranslational regulation of ASBT and will offer new insights into cholangiocyte function and for the understanding of cholestatic liver diseases, where the cholangiocyte is the principle target.
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