The family of organic anion transport proteins (oatp's) has approximately 20 highly homologous members with overlapping substrate specificities. A subset of 3-4 of these proteins is abundantly expressed in the hepatocyte. A number of recent knockout and pharmacogenomic studies have shown that oatp's in the liver are physiologically important and that disturbed function, often due to failure of the transporter to traffic to the cell surface, can alter drug clearance and also cause drug-induced toxicity. Although these are important observations, they provide little insight into the physiology, cell biology, or mechanisms of oatp function. This has been the focus of our studies using oatp1a1 as a prototypical representative of the oatp family. Over the past funding period, we elucidated physiologically important pathways that regulate oatp1a1 transport function. Specifically, we showed that transport activity of oatp1a1 is down-regulated following phosphorylation at serines 634 and 635. We also showed that most oatp's have a PDZ consensus binding domain, and that oatp1a1 requires interaction with PDZK1, a protein with four independent PDZ consensus binding sites, to reside on the plasma membrane. In the absence of PDZK1, oatp1a1 accumulates in vesicles within the cell, and transport activity by the liver is reduced. In further studies, we showed that PDZK1 can bind to EBP50 (NHERF-1), a protein with two independent PDZ consensus binding sites. Our recent experiments in hepatocytes prepared from wild type and EBP50 knockout mice indicate that interaction of the oatp1a1-PDZK1 complex with EBP50 negatively regulates transport function. The focus of the current proposal is to elucidate the mechanisms by which these factors regulate oatp transport activity and subcellular distribution. The objective of this proposal is to test hypotheses that will permit mechanistic elucidation of physiologically important proteins and factors that regulate the subcellular trafficking of oatp1a1 and that are required for expression of active transporter on the hepatocyte basolateral (sinusoidal) plasma membrane.
The Specific Aims are: (1) To identify and characterize physiologically relevant proteins that interact directly or indirectly with oatp1a1. (2) To test the hypothesis that oatp1a1-based protein-protein interactions regulate its subcellular distribution and transport activity. (3) To utilize novel microscopy-based techniques to discover and examine directly the mechanism and role of vesicular trafficking of oatp1a1 on transport activity and subcellular targeting. The oatp's of the hepatocyte are important determinants of drug disposition, and the proposed studies should provide novel insights into their normal physiologic function, as well as perturbations that may cause disease. The physiologic implications of these studies are significant, as disturbed function of oatp's can have substantial negative clinical sequelae through altered drug clearance and metabolism.
Removal of drugs from the circulation by specific transport proteins and their subsequent metabolism is an important function of the liver. A particular group of transporters, the organic anion transport proteins (oatp's), have been found to be important determinants of drug disposition. Recent studies indicate that when oatp function is perturbed, toxic reactions can result. We have found that the liver cell can regulate the distribution of oatp's between the cell surface and sites within the cell where they are inactive. We hypothesize that some of the unanticipated adverse events in patients may be due to differences in this regulatory mechanism, which is the subject of this proposal.
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