CGMP-LINKED RECEPTORS FOR THE ENTEROTOXIN STA OF E COLI

大肠杆菌肠毒素 STA 的 CGMP 连接受体

• 批准号: 2150153
• 负责人: John K. Crane
• 金额: $ 9.57万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-17 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2150153
• 关键词: Escherichia coli; adduct; antibody; biological signal transduction; chemical binding; crosslink; cyclic GMP; enterotoxins; gene expression; guanylate cyclase; immunocytochemistry; laboratory rabbit; molecular site; phosphorylation; protein kinase C; receptor binding; secretion; site directed mutagenesis; synthetic peptide

Toxigenic E.coli infection is one of the most common causes of childhood diarrhea in developing countries, where it is often accompanied by fatal dehydration, as well as the most common cause of traveler's diarrhea in adults. The heat-stable enterotoxin of E. coli (STa), a small cysteine- rich peptide, triggers fluid and electrolyte secretion by stimulating cyclic GMP. Molecular cloning studies by other investigators have shown that intestinal membrane-bound guanylate cyclase serves as a receptor for STa. Nevertheless, the domain(s) of the receptor important for toxin binding remain unknown. In addition, recent work by the author and collaborators has shown that the toxic actions of STa are enhanced by agents which activate protein kinase C (PKC), including phorbol esters and humoral agents able to activate PKC (carbachol, histamine). The interaction between PKC activators and STa can be observed in intact cells, broken cells, and in membranes treated with highly purified PKC and can be observed as an increase in cyclic GMP production and, in intact cell monolayers, as a marked synergistic chloride secretory response. The implications of these findings are that host susceptibility to the toxin may be modulated by neurohumoral signals in the host. The broad, long-term goals of this project are to understand the nature and function of the STa receptor/guanylate cyclase, and how the cGMP interacts with other signalling systems, especially PKC, in intestinal secretion. Specific aims include: (l) to determine the STa-binding domain or domains on the extracellular portion of intestinal gnanylate cyclase by using biochemical, immunologic, and site-directed mutagenic approaches; and (2) to determine whether the effects of PKC are, as we hypothesize, due to direct phosphorylation of guanylate cyclase, and if so to determine the site(s) of phosphorylation by phosphopeptide mapping and site-directed mutagenesis. The only modern therapeutic advance in the treatment of diarrhea, oral rehydration therapy, was introduced more than twenty years ago following the discovery of glucose-sodium cotransport in basic investigations. A rededication to understanding basic biochemical mechanisms controlling intestinal secretion and cell signalling may yield the clues needed for future advances in therapy which are so widely needed. Just as cholera toxin and pertussis toxin played key roles in elucidating the structure and function of G-proteins and adenylate cyclase the unusual, hormone-like toxin STa will likely lead to new understanding of intestinal guanylate cyclase and its regulation.

毒素大肠杆菌感染是最常见的童年原因之一 发展中国家的腹泻，经常伴有致命 脱水，也是旅行者腹泻的最常见原因 成年人。大肠杆菌（STA）的热稳定肠毒素，一种小半胱氨酸 富含肽，触发流体和电解质分泌 循环GMP。其他研究者的分子克隆研究已经表明 肠膜结合的鸟苷酸环化酶充当 Sta。然而，受体的结构域对毒素很重要 结合仍然未知。此外，作者最近的工作和 合作者表明，STA的有毒作用得到了增强 激活蛋白激酶C（PKC）的药物，包括佛比尔酯和 能够激活PKC的体液剂（Carbachol，组胺）。这 PKC激活剂与Sta之间的相互作用可以完整观察 细胞，破碎的细胞以及用高度纯化的PKC处理的膜中 可以观察到是循环GMP产生的增加，并且完整 细胞单层，是一种明显的协同氯化物分泌反应。这 这些发现的含义是宿主对毒素的敏感性 可以通过宿主中的神经肿瘤信号调节。宽阔，长期 该项目的目标是了解STA的性质和功能 受体/鸟苷酸环化酶，以及CGMP如何与其他 信号系统，尤其是肠道分泌中的PKC。具体目标 包括：（l）确定sta结合域或域上的域 通过使用 生化，免疫和定向的诱变方法； （2） 正如我们假设的那样，确定PKC的影响是否是 鸟苷酸环化酶的直接磷酸化，如果是 通过磷酸化的位点通过磷酸化和定向位置 诱变。 口服腹泻的唯一现代治疗进展 二十多年前引入了补液疗法 在基本研究中发现了葡萄糖 - 钠共转移。一个 重新理解控制基本的生化机制 肠分泌和细胞信号传导可能会产生所需的线索 非常需要的未来治疗进展。 就像霍乱毒素和百日咳毒素在阐明中起关键作用 G蛋白和腺苷酸环化酶的结构和功能 异常的，类似激素的毒素STA可能会导致对 肠鸟苷酸环化酶及其调节。