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个具有重叠底物特异性的高度同源成员。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，燕麦蛋白在肝脏中具有重要的生理意义，其功能障碍通常是由于转运蛋白不能运输到细胞表面，可以改变药物清除，也可以导致药物毒性。尽管这些都是重要的观察结果，但它们对燕麦功能的生理学、细胞生物学或机制提供的洞察很少。这一直是我们以Oatp1a1为典型代表的燕麦家族的研究重点。在过去的资金支持期间，我们阐明了调节Oatp1a1转运功能的重要生理途径。具体地说，我们发现Oatp1a1的转运活性在丝氨酸634和635被磷酸化后下调。我们还发现，大多数燕麦都有一个PDZ共识结合域，而Oatp1a1需要与PDZK1相互作用，PDZK1是一种具有四个独立的PDZ共识结合位点的蛋白质，位于质膜上。在没有PDZK1的情况下，燕麦1a1在细胞内的小泡中积累，肝脏的转运活动减少。在进一步的研究中，我们发现PDZK1可以与EBP50(NHERF-1)结合，EBP50是一种具有两个独立的PDZ共识结合位点的蛋白质。我们最近在野生型和EBP50基因敲除小鼠制备的肝细胞上的实验表明，Oatp1a1-PDZK1复合体与EBP50的相互作用对转运功能具有负面调节作用。当前提案的重点是阐明这些因子调节燕麦的运输活性和亚细胞分布的机制。这项建议的目的是测试假说，以允许机制阐明生理上重要的蛋白质和因子，调节Oatp1a1的亚细胞运输，并在肝细胞基侧(正弦)质膜上表达活性转运蛋白所必需的。其具体目标是：(1)鉴定和鉴定直接或间接与Oatp1a1相互作用的生理相关蛋白。(2)验证基于Oatp1a1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)利用基于显微镜的新技术，直接发现和检测囊泡转运Oatp1a1在转运活性和亚细胞靶向方面的机制和作用。肝细胞的燕麦蛋白是药物处置的重要决定因素，拟议的研究应该为它们的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重大，因为燕麦蛋白功能紊乱可能会通过改变药物清除和代谢而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.