IP3 receptor ubiquitination and down-regulation

IP3受体泛素化和下调

• 批准号: 7106452
• 负责人: RICHARD J H WOJCIKIEWICZ
• 金额: $ 27.38万
• 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 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106452
• 关键词: RNA interference; 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

DESCRIPTION (provided by applicant): The general area I research is the intracellular signaling that occurs in response to cell surface receptor activation. In particular, I study inositol 1,4,5-trisphosphate (IP3) receptors, proteins that form ion channels in endoplasmic reticulum (ER) membranes and which, upon the binding of IP3 and Ca2+, mobilize Ca2+ stored in the ER. My long-term objectives are to understand the role of IP3 receptors in intracellular signaling and the mechanisms by which they are regulated, and the mechanism by which signaling proteins are degraded by the ubiquitin / proteasome pathway (UPP). In recent years I have focused on a phenomenon termed "IP3 receptor down-regulation". This is an adaptation to cell surface receptor stimulation that rapidly reduces cellular IP3 receptor content and, thus, the sensitivity of ER Ca2+ stores to IP3. It results from an acceleration of IP3 receptor degradation via the UPP, the route for destruction of many key proteins. Currently, however, very little is known about the molecular details of IP3 receptor down-regulation. The current proposal is designed to rectify this through three Specific Aims. (1) Identification of the ubiquitin-protein ligase that mediates IP3 receptor ubiquitination. The approach taken will be to define the roles of candidate ligases in ubiquitination by inhibiting their expression using RNA interference (RNAi). (2) Identification of the proteins responsible for coupling ubiquitinated IP3 receptors to the proteasome. The approach taken will be to determine which proteins associate with IP3 receptors as they becomes ubiquitinated and then define their roles in coupling IP3 receptors to the proteasome by inhibiting their expression using RNAi. (3) Definition of the events that trigger UN receptor ubiquitination. The approach taken will be to mutate IP3 receptors and manipulate the levels or activities of intracellular signals to identify factors and regions of IP3 receptors that trigger ubiquitination, and then to examine possible structural changes in these regions. Accomplishment of these Aims will both further our understanding of IP3 receptor down-regulation, and provide a paradigm of how ER proteins are degraded by the UPP in mammalian cells. The health relevance of this work is three fold. First, it will lead to a better understanding of the 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 therapeutic and recreational drugs. Second, IP3 receptor down-regulation occurs in several pathophysiological conditions and, thus, obtaining a deeper understanding of its mechanism is of potential benefit to those suffering from these conditions. Third, because of its pivotal role in controlling cell function, the UPP is being explored as a therapeutic target, particularly in cancer. Obtaining a better understanding of the enzymes and mechanisms used in the UPP will both facilitate drug design and give us a better appreciation of their effects.

描述(申请人提供)：我研究的一般领域是响应细胞表面受体激活而发生的细胞内信号。特别是，我研究了肌醇1，4，5-三磷酸(IP3)受体，这是一种在内质网(ER)膜上形成离子通道的蛋白质，当IP3与钙结合时，它可以动员储存在内质网中的钙离子。我的长期目标是了解IP3受体在细胞内信号传递中的作用和调控机制，以及泛素/蛋白酶体途径(UPP)降解信号蛋白的机制。近年来，我主要关注一种名为“IP3受体下调”的现象。这是对细胞表面受体刺激的一种适应，这种刺激会迅速减少细胞内IP3受体的含量，从而使内质网钙离子对IP3的敏感性增加。它是通过UPP加速IP3受体降解的结果，UPP是破坏许多关键蛋白质的途径。然而，目前对IP3受体下调的分子细节知之甚少。目前的提案旨在通过三个具体目标纠正这一问题。(1)鉴定介导IP3受体泛素化的泛素蛋白连接酶。所采取的方法将是通过使用RNA干扰(RNAi)抑制候选连接酶的表达来确定它们在泛素化中的作用。(2)泛素化IP3受体与蛋白酶体偶联蛋白的鉴定。所采取的方法将是确定当IP3受体变得泛素化时与它们相关的蛋白质，然后通过使用RNAi抑制IP3受体的表达来确定它们在将IP3受体与蛋白酶体偶联方面的作用。(3)触发UN受体泛素化的事件的定义。采取的方法将是突变IP3受体并操纵细胞内信号的水平或活性，以确定触发泛素化的IP3受体的因素和区域，然后检查这些区域可能的结构变化。 这些目标的实现将加深我们对IP3受体下调的理解，并为哺乳动物细胞中ER蛋白如何被UPP降解提供一个范例。这项工作的健康相关性有三个方面。首先，它将导致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改以及对治疗和娱乐药物效果的耐受性的基础。其次，IP3受体下调发生在几种病理生理条件下，因此，深入了解其机制对那些患有这些疾病的人有潜在的好处。第三，由于其在控制细胞功能方面的关键作用，人们正在探索将UPP作为治疗靶点，特别是在癌症中。更好地了解UPP中使用的酶和机制将有助于药物设计，并使我们更好地了解它们的效果。