IP3 receptor ubiquitination and down-regulation

描述（由申请人提供）：一般领域I研究的是响应于细胞表面受体活化而发生的细胞内信号传导。特别是，我研究肌醇1，4，5-三磷酸（IP 3）受体，蛋白质，形成离子通道在内质网（ER）膜，并在IP 3和Ca 2+的结合，动员Ca 2+存储在ER。我的长期目标是了解IP 3受体在细胞内信号传导中的作用及其调节机制，以及信号蛋白通过泛素/蛋白酶体途径（UPP）降解的机制。近年来，我一直专注于一种称为“IP 3受体下调”的现象。这是对细胞表面受体刺激的适应，其快速降低细胞IP 3受体含量，从而降低ER Ca 2+储存对IP 3的敏感性。它是通过UPP加速IP 3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IP 3受体下调的分子细节知之甚少。目前的建议旨在通过三个具体目标纠正这一点。(1)介导IP 3受体泛素化的泛素-蛋白连接酶的鉴定。所采取的方法将是通过使用RNA干扰（RNAi）抑制其表达来确定候选连接酶在泛素化中的作用。(2)鉴定负责将泛素化IP 3受体偶联至蛋白酶体的蛋白质。所采取的方法将是确定哪些蛋白质与IP 3受体相关，因为它们变得泛素化，然后通过使用RNAi抑制它们的表达来定义它们在将IP 3受体偶联到蛋白酶体中的作用。(3)触发UN受体泛素化的事件的定义。所采取的方法将是突变IP 3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IP 3受体的因子和区域，然后检查这些区域中可能的结构变化。 这些目标的实现将进一步加深我们对IP 3受体下调的理解，并提供一个在哺乳动物细胞中UPP如何降解ER蛋白的范例。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于适应细胞外刺激的机制;这种适应是许多细胞功能生理学修饰和对治疗性和娱乐性药物的耐受性的基础。第二，IP 3受体下调发生在几种病理生理条件下，因此，获得对其机制的更深入了解对那些患有这些条件的人有潜在的益处。第三，由于其在控制细胞功能中的关键作用，UPP正在被探索作为治疗靶点，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作用。

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.