Trafficking and Regulation of Drug/Xenobiotic Transporter OAT

描述（由申请人提供）：有机阴离子转运蛋白（OAT）介导多种环境毒素和临床重要药物（包括抗HIV治疗剂、抗肿瘤药物、抗生素、抗高血压药和抗炎药）的吸收、分布和排泄。OAT主要在肾脏、肝脏、脑和胎盘中表达。这些器官中的OAT功能障碍显著导致肾、肝、神经和胎儿毒性和疾病。我们的长期目标是确定通过OAT途径的药物处置的分子机制。在上一个赠款期间，取得了重大进展和生产力，这一时期的新发现导致在这一竞争性更新中建立了一个微调的研究计划和战略。我们建议测试新的假设，Nedd 4 -2，泛素连接酶，作为一个中心的会聚点/开关的蛋白激酶调节OAT 1的活动，因此，用于转导不同的生理刺激OAT 1介导的药物转运。提出了三个具体目标。在特定目标I中，我们将绘制Nedd 4 -2上蛋白激酶特异性磷酸化位点，并研究Nedd 4 -2的磷酸化是否是不同蛋白激酶调节OAT 1转运活性的机制。在具体目标II中，我们将确定具体的 OAT 1和Nedd 4 -2中的结构域/氨基酸残基，对这两种蛋白质之间的相互作用至关重要。在Specific Aim III中，我们将评估Nedd 4 -2在OAT 1介导的药物转运中的生理作用。生物化学和分子生物学的组合方法将用于培养细胞以及正常和Nedd 4 -2敲除小鼠的肾切片中的拟议研究。了解Nedd 4 -2的动态磷酸化在OAT调控中的作用，这是药物转运领域的一个新焦点，将对未来设计旨在最大化治疗效果和最小化毒性的策略产生重大影响，并将允许深入了解肾脏，肝脏，神经学和胎儿毒性和疾病的分子，细胞和临床基础。

DESCRIPTION (provided by applicant): Organic anion transporters (OATs) mediate the absorption, distribution, and excretion of a diverse array of environmental toxins, and clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. OATs are mainly expressed in the kidney, liver, brain and placenta. OAT dysfunction in these organs significantly contributes to the renal, hepatic, neurological and fetal toxicity and disease. Our long-term goal is to define the molecular mechanisms underlying drug disposition through the OAT pathway. During the previous grant period, significant progress and productivity have been achieved, and the new findings from this period led to the establishment of a fine-tuned research plan and strategy in this competing renewal. We propose to test the novel hypothesis that Nedd4-2, an ubiquitin ligase, serves as a central convergence point/switch for protein kinase-regulated OAT1 activity, and therefore for transducing diverse physiological stimuli to OAT1-mediated drug transport. Three Specific Aims are outlined. In Specific Aim I, we will map protein kinase-specific phosphorylation sites on Nedd4-2, and examine whether phosphorylation of Nedd4-2 is the mechanism by which diverse protein kinases regulate OAT1 transport activity. In Specific Aim II, we will identify the specific domains/amino acid residues in OAT1 and Nedd4-2, critical for the interaction between these two proteins. In Specific Aim III, we will evaluate the physiological role of Nedd4-2 in OAT1-mediated drug transport. Combined approaches of biochemistry and molecular biology will be employed for the proposed studies in cultured cells, and in kidney slices from normal and Nedd4-2 knockout mice. Understanding the role of dynamic phosphorylation of Nedd4-2 in the regulation of OATs, a novel focus in drug transport field, will have significant impact on the future design of strategies aimed at maximizing therapeutic efficacy and minimizing toxicity, and will permit insight into the molecular, cellular, and clinical bases of renal, hepatic, neurologica and fetal toxicity and disease.