INSP3 RECEPTOR UBIQUITINATION AND DOWN-REGULATION

INSP3 受体泛素化和下调

• 批准号: 7020908
• 负责人: RICHARD J H WOJCIKIEWICZ
• 金额: $ 5.62万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020908
• 关键词: active sites; biological signal transduction; calcium flux; cell surface receptors; complementary DNA; endoplasmic reticulum; inositol phosphates; intracellular transport; laboratory rat; proteasome; proteolysis; receptor binding; receptor expression; transfection; ubiquitin

The focus of my research in recent years has been the intracellular signaling that occurs in response to cell surface receptor activation. In particular, I have studied inositol 1, 4, 5-trisphosphate (InsP3) receptors, proteins that form channels in endoplasmic reticulum (ER) membranes and which, in response to InsP3 binding, mobilize Ca2+ stored within the ER. My long-term objectives are (i) to understand the role of InsP3 receptors in intracellular signaling, (ii) to define mechanisms that regulate InsP3 receptors and other signaling proteins, and (iii) to establish the biological significance of InsP3 receptor regulation. Since 1991, I have studied InsP3 receptor down-regulation, a novel adaptive response to cell surface receptor activation that rapidly reduces cellular InsP3 receptor content and, thus, the sensitivity of ER Ca2+ stores to InsP3. Recently, it has been shown that InsP3 receptor down-regulation is mediated by the ubiquitin / proteasome pathway, a crucial pathway for the degradation of many cellular proteins that is currently being considered as a therapeutic target. Ubiquitination of InsP3 receptors is the event that initiates their degradation. The Specific Aims of the current proposal are (1) to define the site(s) of ubiquitination in InsP3 receptors using biochemical techniques and InsP3 receptor mutagenesis, (2) to identify the enzymes responsible for InsP3 receptor ubiquitination using biochemical techniques and transfection of cDNAs encoding these enzymes, (3) to define the signaling events that accelerate InsP3 receptor ubiquitination using InsP3 receptor mutagenesis, and (4) to characterize InsP3 receptor ubiquitination and down-regulation in rat brain. Accomplishment of these Aims will define the events that cause InsP3 receptor ubiquitination and will provide information on it's biological significance. The health relevance of this work is threefold. First, it will lead to a better understanding of mechanisms that cells use to adapt to extracellular stimuli; such adaptation is the basis for many physiological modifications to cell function and of tolerance to the effects of many therapeutic and recreational drugs. Second, it will establish whether muscarinic agonists used in the treatment of Alzheimer's disease cause InsP3 receptor ubiquitination and down-regulation in vivo. Third, it will help to map the ubiquitin / proteasome pathway and, thus, will provide a better understanding of drugs designed to interfere with this process.

近年来我的研究重点是细胞表面受体激活后发生的细胞内信号转导。特别是，我研究了肌醇1,4,5 -三磷酸（InsP3）受体，这是一种在内质网（ER）膜上形成通道的蛋白质，它在与InsP3结合时调动内质网内储存的Ca2+。我的长期目标是(i)了解InsP3受体在细胞内信号传导中的作用，（ii）定义调节InsP3受体和其他信号蛋白的机制，（iii）建立InsP3受体调节的生物学意义。自1991年以来，我研究了InsP3受体下调，这是一种对细胞表面受体激活的新型适应性反应，可迅速降低细胞中InsP3受体的含量，从而降低ER Ca2+储存对InsP3的敏感性。最近，研究表明，InsP3受体下调是由泛素/蛋白酶体途径介导的，泛素/蛋白酶体途径是许多细胞蛋白降解的关键途径，目前被认为是治疗靶点。InsP3受体的泛素化是引发其降解的事件。本研究的具体目标是：(1)利用生化技术和InsP3受体诱变来确定InsP3受体泛素化位点；(2)利用生化技术和转染编码这些酶的cdna来确定负责InsP3受体泛素化的酶；(3)利用InsP3受体诱变来确定加速InsP3受体泛素化的信号事件。(4)观察大鼠脑中InsP3受体泛素化及下调的特征。完成这些目标将定义导致InsP3受体泛素化的事件，并将提供有关其生物学意义的信息。这项工作的健康相关性是三重的。首先，它将使我们更好地理解细胞用来适应细胞外刺激的机制；这种适应是对细胞功能的许多生理修饰和对许多治疗性和娱乐性药物作用的耐受性的基础。第二，确定用于治疗阿尔茨海默病的毒蕈碱激动剂是否在体内引起InsP3受体泛素化和下调。第三，它将有助于绘制泛素/蛋白酶体途径，从而更好地理解设计用于干扰这一过程的药物。