INSP3 RECEPTOR UBIQUITINATION AND DOWN-REGULATION

INSP3 受体泛素化和下调

• 批准号: 6043439
• 负责人: RICHARD J H WOJCIKIEWICZ
• 金额: $ 21.28万
• 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 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043439
• 关键词: active sites; biological signal transduction; calcium flux; complementary DNA; endoplasmic reticulum; inositol phosphates; intracellular transport; laboratory rat; proteasome; proteolysis; receptor; 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响应细胞表面受体激活而发生的细胞内信号传导。 特别是，我研究了肌醇 1,4,5-三磷酸 (InsP3) 受体，这种蛋白质在内质网 (ER) 膜中形成通道，并响应 InsP3 结合，动员储存在 ER 内的 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，以及 (iii) 确定 InsP3 受体调节的生物学意义。自 1991 年以来，我一直在研究 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 InsP3 受体含量，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，这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。 当前提案的具体目标是 (1) 使用生化技术和 InsP3 受体诱变来定义 InsP3 受体泛素化位点，(2) 使用生化技术和编码这些酶的 cDNA 转染来识别负责 InsP3 受体泛素化的酶，(3) 定义加速 InsP3 受体的信号传导事件 使用 InsP3 受体诱变进行泛素化，以及 (4) 表征大鼠脑中 InsP3 受体泛素化和下调的特征。这些目标的实现将确定导致 InsP3 受体泛素化的事件，并提供有关其生物学意义的信息。 这项工作与健康的相关性有三个方面。 首先，它将有助于更好地理解细胞适应细胞外刺激的机制；这种适应是细胞功能的许多生理改变以及对许多治疗和娱乐药物作用的耐受性的基础。 其次，将确定用于治疗阿尔茨海默病的毒蕈碱激动剂是否会导致体内InsP3受体泛素化和下调。第三，它将有助于绘制泛素/蛋白酶体途径图谱，从而使人们更好地了解旨在干扰这一过程的药物。