The hepatocyte plays an essential role in clearing drugs and endogenous compounds, many of which are organic anions, from the circulation. Our focus is on two families of transport proteins that mediate hepatocyte organic anion uptake: the organic anion transport proteins (OATPs) and the Na+-taurocholate cotransporting polypeptide (NTCP). They are normally localized to the plasma membrane as well as intracellular vesicles. At steady state, there is a balance between inward and outward movement of these vesicles such that plasma membrane localization predominates. Disruption of this equilibrium can upset this balance, resulting in altered plasma membrane expression. Only those transporters expressed on the hepatocyte surface can mediate uptake. Reduced surface localization has been associated with elevated circulating levels of drugs and concomitant toxicity. The overall goal of this proposal is elucidation of these novel trafficking mechanisms as they relate to liver function in health and disease. The current proposal addresses two important aspects of vesicle targeting as they relate to transporter subcellular location and activity: the molecular basis of intracellular motility and trafficking of transporter-containing vesicles; and the functional role f proteins that recruit specific motors and accessory proteins to transporter-containing vesicles To this end we will: 1) define the role of specific PDZ proteins in recruiting motor and regulatory proteins to OATP-containing vesicles; 2) elucidate the mechanism that mediates trafficking to the plasma membrane of OATPs lacking a PDZ consensus binding motif; and 3) characterize mechanisms regulating the subcellular distribution of ntcp. We will use novel procedures and technology that we have developed and validated to study mechanisms of microtubule-based transporter-containing vesicle motility and trafficking in cell lines and in rat, mouse, and human liver. These technologies include use of an in vitro microtubule-based motility system; preparation of endocytic vesicles from cell lines and intact liver that retain associated motors and regulatory proteins; analysis of vesicle fissioning in vitro as an indicator of segregation of transport proteins; and use of sorting technology to purify specific vesicle populations that can be subjected to proteomic analysis. The major significance of the proposed studies is the potential for development of new insight into the molecular mechanisms that govern transporter trafficking and surface expression in hepatocytes. It is possible that mutation of a single accessory protein could affect activity of several transporters that it regulates. Successful completion of these studies may lead to new paradigms governing liver-mediated clearance of drugs from the circulation and may provide new, previously unexpected targets for consideration in pharmacogenetic profiling of individual patients.
The liver is essential to removal of many drugs and toxins from the circulation, using specific proteins (transporters) that are in equilibrium between the cel surface and vesicles within the cell. Disruption of this equilibrium can result in lack of transporer at the cell surface and consequent drug toxicity. This proposal will examine the molecular mechanisms that govern transporter surface expression in hepatocytes and may provide new insight into toxic effects of drugs that are normally cleared by the liver.
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