MECHANISM OF ACTION OF VASOPRESSIN

加压素的作用机制

• 批准号: 6177285
• 负责人: DENNIS A AUSIELLO
• 金额: $ 45.65万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177285
• 关键词: G protein; MDCK cell; Xenopus oocyte; biological signal transduction; biotin; caveolins; clathrin; confocal scanning microscopy; dynamin; electrophysiology; endocytosis; fluorescent dye /probe; green fluorescent proteins; hormone receptor; immunocytochemistry; intracellular transport; membrane activity; molecular cloning; neuropeptide receptor; protein structure function; receptor coupling; receptor expression; receptor sensitivity; sodium channel; surface plasmon resonance; vasopressins

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.

这项建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， 血管加压素，它在调节盐和水方面起着重要作用 通过对肾脏的作用来平衡。 它的功能障碍是 先天性肾源性尿崩症（CNDI）和继发性 液体和电解质异常的原因，如 充血性心力衰竭、肝硬化和肾病综合征。 本提案的初步目的是分析 加压素V2受体（V2 R）的结构基序负责 1)在内质网和高尔基体中正确的折叠和加工， 靶向肾细胞的基底外侧（或顶端）膜;和2） 调节脱敏、下调或再敏感的 我们提出的V2 R是由G蛋白受体激酶介导的 磷酸化、抑制蛋白结合、内吞和胞吐 由网格蛋白包被的凹坑或小窝引发。 我们还建议， 这些事件中的至少一个由异源三聚体G蛋白协调 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V2 R或其突变、CNDI突变体，或 “分裂”V2 R，通过免疫细胞化学、共聚焦显微镜和免疫组织化学来监测这些过程。 和电子显微镜或细胞表面生物素化在固定和活 细胞 V2 R与类似标记的辅助蛋白的关联 (e.g. G/alpha/i3，网格蛋白和衔接蛋白，发动蛋白或小窝蛋白） 将通过免疫细胞化学和免疫沉淀或通过 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 在其生理作用中的元素也需要G/α/i3调节 加压素敏感性Na+通道，称为5 pS（rENaC） 或9 pS通道。 我们建议的最终目的是确定 G/alpha/i3调节5 pS rENaC的能力，通过 电生理技术在卵母细胞或共表达 rENaC和组成型活性、百日咳毒素不敏感的G/α/i3 （G/alpha/i3* PT neg）。 将进行研究以确定G/α/i3 作用取决于其先前确定的膜靶向序列 在我们的实验室里。 直接示范的行动 G/alpha/i3*PTneg在5 pS和9 pS Na+通道上的突变将 在加入以下物质后，通过电生理技术测定 将纯化的蛋白质结合到A6或A6细胞膜的胞质表面， 表达rENaC的MDCK细胞或MDCK细胞，或加入纯化的 牛肾9 pS Na+通道重建成脂质双层。 最后，利用经由六组氨酸偶联的G/alpha/13 *PTneg， 镍-琼脂糖珠，G/α/i3对Na+作用的效应蛋白 通道将被表达克隆。