Hepatic Organic Anion Uptake and Transport

描述（由申请人提供）：有机阴离子转运蛋白家族（oatp’s）大约有20个高度同源的成员，它们具有重叠的底物特异性。这些蛋白中的3-4个亚群在肝细胞中大量表达。最近的一些基因敲除和药物基因组学研究表明，肝脏中的oatp在生理上是重要的，而功能紊乱（通常是由于转运体无法运输到细胞表面）可以改变药物清除并引起药物诱导的毒性。尽管这些都是重要的观察结果，但它们对oatp功能的生理学、细胞生物学或机制提供的见解很少。这是我们以oatp1a1作为oatp家族的典型代表进行研究的重点。在过去的资助期内，我们阐明了调节oatp1a1转运功能的生理重要途径。具体来说，我们发现oatp1a1的转运活性在634和635丝氨酸磷酸化后下调。我们还发现，大多数oatp具有PDZ一致结合域，并且oatp1a1需要与PDZK1相互作用才能在质膜上存在，PDZK1是一种具有四个独立PDZ一致结合位点的蛋白质。在缺乏PDZK1的情况下，oatp1a1在细胞内的囊泡中积累，肝脏的转运活性降低。在进一步的研究中，我们发现PDZK1可以结合EBP50 (NHERF-1)， EBP50是一种具有两个独立PDZ共识结合位点的蛋白质。我们最近在野生型和EBP50敲除小鼠制备的肝细胞中进行的实验表明，oatp1a1-PDZK1复合物与EBP50的相互作用负性调节转运功能。本研究的重点是阐明这些因子调控oatp转运活性和亚细胞分布的机制。本提案的目的是测试假设，这些假设将允许对调节oatp1a1亚细胞运输的生理上重要的蛋白质和因子的机制阐明，这些蛋白质和因子是肝细胞基底外侧（窦状）质膜上活性转运蛋白表达所必需的。具体目的是：(1)鉴定和表征与oatp1a1直接或间接相互作用的生理相关蛋白。(2)验证基于oatp1a1的蛋白-蛋白相互作用调节其亚细胞分布和转运活性的假说。(3)利用新的基于显微镜的技术，直接发现和研究oatp1a1在转运活性和亚细胞靶向中的囊泡转运机制和作用。肝细胞的oatp是药物处置的重要决定因素，拟议的研究应该为其正常生理功能以及可能导致疾病的扰动提供新的见解。这些研究的生理学意义是显著的，因为oatp的功能紊乱可以通过改变药物清除和代谢而产生大量负面的临床后遗症。

DESCRIPTION (provided by applicant): 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.