MECHANISM OF ACTION OF VASOPRESSIN

这项提议的长期目标是提供一个基本的 了解神经垂体激素的细胞生物学， 加压素，它在盐和水的调节中起着重要作用 通过对肾脏的作用达到平衡。它的功能障碍是一个主要的 先天性肾源性尿崩症的病因和继发性尿崩症 引起液体和电解质异常的疾病 充血性心力衰竭、肝硬变和肾病综合征。 这项建议的最初目标是提供对 加压素V2受体(V2R)的结构基序负责 1)在ER和高尔基体中正确折叠和加工，并适当 靶向肾细胞的基侧(或顶端)膜；2) 对脱敏、下调或重新增敏的调节 我们提出的V2R是由G蛋白受体激酶介导的 磷酸化、arrestin结合、内吞和胞吐 由包被网状蛋白的凹坑或凹陷引发。我们还提出了一个 或更多的这些事件是由异源三聚体G蛋白协调的 亚基G/α/I3。这些研究将利用表位标记或 绿色荧光蛋白标记的V2Rs或其突变、CNDI突变体或 “分裂”的V2Rs通过免疫细胞化学、共聚焦来监测这些过程 和电子显微镜或细胞表面生物素化在固定和活的 细胞。V2R与相似标记辅助蛋白的结合 (例如G/α/I3、网状蛋白和接头蛋白、动力素或小窝蛋白) 将通过免疫细胞化学和免疫沉淀或由 表面等离子体共振的新方法。 加压素受体信号通路向远端的转导 其生理作用中的元素也需要G/α/I3调节 对加压素敏感的Na+通道，称为5pS(RENaC) 或9 ps通道。我们提案的最终目标是确定 G/α/I3调节5ps rENaC的能力，由 卵母细胞或MDCK细胞共表达的电生理技术 RENaC和对百日咳毒素不敏感的活性成分G/α/I3 (G/Alpha/I3*PTneg)。将进行研究以确定G/α/I3 作用取决于先前确定的ITS膜靶向序列 在我们的实验室里。行为的直接演示 G/α/I3*PTneg在5pS和9pS钠离子通道上的突变将 由电生理技术在添加后确定 纯化的蛋白进入A6细胞膜的胞浆表面 表达rENaC的细胞或MDCK细胞，或加入纯化的 牛肾9PS钠离子通道重组为脂双层。 最后，利用经六氢异硫氨酸偶联的G/α/I3*PTneg 镍-琼脂糖珠，G/α/I3对Na+作用的效应蛋白 将对通道进行表达克隆。

The long-term objective of this proposal is to provide a fundamental understanding of the cell biology of the neurohypophyseal hormone, vasopressin, which plays a major role in the regulation of salt and water balance through its action on the kidney. Its dysfunction is a primary cause of congenital nephrogenic diabetes insipidus (CNDI) and a secondary cause of fluid and electrolyte abnormalities in such diseases as congestive heart failure, cirrhosis of the liver, and nephrotic syndrome. The initial aims of this proposal are to provide an analysis of the structural motifs of the vasopressin V2 receptor (V2R) responsible for 1) proper folding and processing in the ER and Golgi and appropriate targeting to basolateral (or apical) membranes of kidney cells; and 2) regulation of desensitization, downregulation or resensitization of the V2R that we propose is mediated by G protein receptor kinase phosphorylation, arrestin binding, and endocytosis and exocytosis initiated by clathrin-coated pits or caveolae. We also propose that one or more of these events is coordinated by the heterotrimeric G protein subunit, G/alpha/i3. These studies will make use of epitope-tagged or green fluorescent protein-tagged V2Rs or its mutations, CNDI mutants, or "split" V2Rs to monitor these processes by immunocytochemistry, confocal and electron microscopy or cell surface biotinylation in fixed and living cells. Association of the V2R with similarly tagged auxiliary proteins (e.g. G/alpha/i3, clathrin and adaptor proteins, dynamin or caveolin) will be determined by immunocytochemistry and immunoprecipation or by a new method of surface plasmon resonance. Transduction of the vasopressin receptor signaling pathway to the distal elements in its physiologic action also requires G/alpha/i3 regulation of vasopressin-sensitive Na+ channels, referred to as the 5 pS (rENaC) or the 9 pS channel. The final aim of our proposal is to determine the ability of G/alpha/i3 to regulate the 5 pS rENaC, evaluated by electrophysiological techniques in oocytes or MDCK cells co-expressing rENaC and a constitutively active, pertussis-toxin insensitive G/alpha/i3 (G/alpha/i3*PTneg). Studies will be conducted to determine if G/alpha/i3 action depends on its membrane targeting sequences previously determined in our laboratory. Direct demonstration of the action of G/alpha/i3*PTneg on its mutations on the 5 pS and 9 pS Na+ channels will be determined by electrophysiological techniques after the addition of the purified protein to the cytosolic surface of membranes of either A6 cells or MDCK cells expressing rENaC, or following addition to purified bovine renal 9 pS Na+ channels reconstituted into lipid bilayers. Finally, utilizing G/alpha/i3*PTneg coupled via a hexahistidine to nickel-agarose beads, effector proteins for G/alpha/i3 action on Na+ channels will be expression cloned.