Hepatic Organic Anion Uptake and Transport

描述（由申请人提供）：有机阴离子转运蛋白（oatp）家族约有20个高度同源的成员，具有重叠的底物特异性。这些蛋白质中的3-4个亚组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，oatp在肝脏中具有重要的生理学意义，并且通常由于转运蛋白无法运输到细胞表面而导致的功能紊乱可以改变药物清除率，并且还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的生理学、细胞生物学或机制提供的见解很少。这一直是我们使用oatp 1a 1作为oatp家族的典型代表进行研究的焦点。在过去的资助期间，我们阐明了生理上重要的途径，调节oatp 1a 1运输功能。具体来说，我们发现oatp 1a 1的转运活性在丝氨酸634和635处磷酸化后下调。我们还表明，大多数oatp的PDZ共识结合域，和oatp 1a 1需要与PDZK 1，一个蛋白质与四个独立的PDZ共识结合位点，驻留在质膜上的相互作用。在缺乏PDZK 1的情况下，oatp 1a 1在细胞内的囊泡中积累，并且肝脏的运输活性降低。在进一步的研究中，我们发现PDZK 1可以与EBP 50（NHERF-1）结合，EBP 50是一种具有两个独立PDZ共有结合位点的蛋白质。我们最近在野生型和EBP 50基因敲除小鼠肝细胞中的实验表明，oatp 1a 1-PDZK 1复合物与EBP 50的相互作用负调节转运功能。目前建议的重点是阐明这些因子调节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，将允许机制阐明生理上重要的蛋白质和因子，调节oatp 1a 1的亚细胞运输和肝细胞基底外侧（正弦）质膜上的活性转运蛋白的表达所需的。具体目的是：（1）鉴定和表征与oatp 1a 1直接或间接相互作用的生理相关蛋白。(2)验证基于oatp 1a 1的蛋白质-蛋白质相互作用调节其亚细胞分布和转运活性的假设。(3)利用新的显微镜技术直接发现和检测oatp 1a 1的囊泡运输在转运活性和亚细胞靶向中的机制和作用。肝细胞的oatp是药物处置的重要决定因素，拟议中的研究将提供对它们正常生理功能的新见解，以及可能导致疾病的扰动。这些研究的生理学意义是重要的，因为oatp的功能紊乱可能通过改变药物清除和代谢产生实质性的负面临床后遗症。

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.