MOLECULAR PHYSIOLOGY OF UREA TRANSPORTERS

在哺乳动物中，尿素是氮代谢的主要最终产物， 在尿液浓缩机制中起着核心作用。规管 尿素在肾髓质中的排泄和积聚取决于 加压素敏感、根素可抑制尿素的功能状态 传送者。我们最近分离出了编码尿素的cDNA。 兔(UT2)和大鼠肾髓质转运蛋白(RUT2)以及 来自人类红细胞(HUT3)。这项建议的目的是 阐明尿素的结构、功能及其表达调控 传送者。以确定其主要序列中负责的区域 对尿素转运、根皮素的抑制和对汞的敏感性 试剂我们建议通过基因工程来修饰这些蛋白质，并 研究突变蛋白在非洲爪哇卵母细胞中的表达。我们建议 在UT2和HUT3之间构建嵌合体，以分配功能 UT2和HUT3特定区域的属性以及站点定向使用的属性 突变以定位参与被动反应的单个氨基酸残基 尿素运输。为了模拟尿素结合位点，我们建议使用一个 涉及毛细管电泳法的新方法将使我们能够 确定特定突变对底物特异性的影响。一个 利用半胱氨酸反应性的扫描突变程序 放置在分子内的关键位置的PCMBS将用于 探索这些蛋白的转位途径。要研究二次 尿素转运体的结构我们将尝试开发一种 高效表达系统在大肠杆菌中的表达及纯化和重组 用于光谱分析和进行结晶试验的蛋白质。至 确定加压素是否诱导肾脏转运的激活 涉及从细胞内囊泡池中招募尿素转运体 我们建议研究rUT2在终末IMCD细胞中的表达。 细胞内小泡的免疫金电镜法测定 加压素的急性治疗是否会触发rUT2插入 质膜中的分子。因为我们证明了rUT2基因 水平对水合状态的变化有很高的反应 我们建议通过动物或蛋白质饮食控制来确定 引起这些反应的介体并研究其机制 2.9kb和4.0kb转录本的潜在差异调控 RUT2。这些研究的结果应该会促进我们对 哺乳动物能够浓缩尿液的机制应该提供 对我们理解如何促进 溶质转运体起作用。

In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary concentrating mechanism. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of vasopressin-sensitive, phloretin-inhibitable urea transporters. We have recently isolated cDNAs encoding the urea transporters from rabbit (UT2) and rat kidney medulla (rUT2) as well as from human erythrocytes (HUT3). The objective Of this proposal is to elucidate the structure, function and regulation of expression of urea transporters. To identify regions in their primary sequences responsible for urea translocation, phloretin inhibition and sensitivity to mercurial reagents we propose to modify these proteins by genetic engineering and to study the mutant proteins by expression in Xenopus oocytes. We propose to construct chimeras between UT2 and HUT3 in order to assign functional properties to specific regions of UT2 and HUT3 and to used site-directed mutagenesis to pinpoint individual amino acid residues involved in passive urea transport. To model the urea binding site we propose to employ a novel approach involving capillary electrophoresis which will allow us to determine the effect of specific mutations on substrate-specificity. A scanning mutagenesis program that exploits the reactivity of cysteines placed at strategic locations within the molecule to PCMBS will be used to probe the translocation pathway of these proteins. To study the secondary structure of urea transporters we will attempt to develop an overexpression system in E.coli and use the purified and reconstituted protein for spectroscopy and to perform crystallization trials. To determine whether vasopressin-induced activation of transport in kidney involves recruitment of urea transporters from intracellular vesicle pools of terminal IMCD cells we propose to study the expression of rUT2 in intracellular vesicles by immunogold electronmicroscopy and to determine whether acute treatment with vasopressin triggers insertion of rUT2 molecules in the plasma membrane. Since we demonstrated that rUT2 mRNA levels are highly responsive to changes in the hydration state of the animal or to protein dietary manipulations we propose to determine the mediators which cause these responses and to study the mechanism underlying differential regulation of the 2.9 and 4.0 kb transcripts of rUT2. The results from these studies should advance our understanding of the mechanisms that enable mammals to concentrate urine and should provide information of general importance to our understanding of how facilitated solute transporters function.