Trafficking and Regulation of Drug/Xenobiotic Transporter OAT

描述（由申请人提供）：有机阴离子转运体（OATs）介导多种环境毒素和临床重要药物的吸收、分布和排泄，包括抗hiv治疗药物、抗肿瘤药物、抗生素、抗高血压药和抗炎药。燕麦主要在肾、肝、脑和胎盘中表达。这些器官的OAT功能障碍显著地导致肾、肝、神经和胎儿的毒性和疾病。我们的长期目标是通过OAT途径确定药物处置的分子机制。在上一个资助期内，我们取得了重大进展和生产力，这一期间的新发现促使我们在这次竞争性更新中制定了一项微调的研究计划和战略。我们提出验证一种新的假设，即Nedd4-2，一种泛素连接酶，作为蛋白激酶调节的OAT1活性的中心汇聚点/开关，从而将各种生理刺激转导到OAT1介导的药物运输。概述了三个具体目标。在Specific Aim I中，我们将在Nedd4-2上绘制蛋白激酶特异性磷酸化位点，并研究Nedd4-2的磷酸化是否是多种蛋白激酶调节OAT1转运活性的机制。在具体目标II中，我们将确定具体目标

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.