MOLECULAR PHYSIOLOGY OF UREA TRANSPORTERS

在哺乳动物中，尿素是氮代谢的主要终产物， 在尿浓缩机制中起着核心作用。调控 肾髓质中的尿素排泄和积聚取决于 加压素敏感性、根皮素敏感性尿素的功能状态 运输机 我们最近分离出编码尿素的cDNA， 来自兔（UT2）和大鼠肾髓质（rUT2）的转运蛋白以及 来自人红细胞（HUT 3）。本提案的目的是 阐明尿素的结构、功能及表达调控 运输机以确定其一级序列中负责 对尿素转运、根皮素抑制和对汞的敏感性 我们建议通过基因工程来修饰这些蛋白质， 研究突变蛋白在非洲爪蟾卵母细胞中的表达。我们建议 在UT2和HUT3之间构建嵌合体，以分配功能性 UT2和HUT3的特定区域的特性以及所使用的定点 诱变以精确定位参与被动免疫应答的单个氨基酸残基。 尿素转运为了模拟尿素结合位点，我们建议采用 一种新的毛细管电泳方法， 确定特定突变对底物特异性的影响。一 利用半胱氨酸的反应性的扫描诱变程序 放置在分子内的战略位置，以PCMBS将用于 探索这些蛋白质的转运途径。学习中学 尿素转运蛋白的结构，我们将尝试开发一种 在大肠杆菌中的过表达系统，并使用纯化和重构的 用于光谱分析和进行结晶试验的蛋白质。到 确定加压素是否诱导肾脏中转运的激活 包括从胞内囊泡库募集尿素转运蛋白 我们建议研究终末IMCD细胞中rUT 2的表达， 免疫金电子显微镜检查细胞内囊泡，并确定 加压素急性治疗是否触发rUT2插入 细胞膜中的分子。 由于我们证明了rUT2 mRNA 水平是高度响应于水化状态的变化， 动物或蛋白质饮食操作，我们建议确定 介体引起这些反应，并研究其机制， 潜在的差异调节的2.9和4.0 kb的转录本， rUT 2.这些研究的结果应该会促进我们对 使哺乳动物能够浓缩尿液的机制， 对我们理解如何促进 溶质转运蛋白的功能。

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.