IP3 receptor ubiquitination and down-regulation

描述（由申请人提供）：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传导。我特别研究了肌醇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，在IP3和Ca2+结合后，可以调动储存在内质网中的Ca2+。我的长期目标是了解IP3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，我一直关注一种被称为“IP3受体下调”的现象。这是一种对细胞表面受体刺激的适应，可以迅速降低细胞IP3受体含量，从而降低ER Ca2+储存对IP3的敏感性。它是通过UPP加速IP3受体降解的结果，UPP是许多关键蛋白质破坏的途径。然而，目前对IP3受体下调的分子细节知之甚少。目前的建议旨在通过三个具体目标来纠正这一问题。(1)介导IP3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)鉴定了泛素化IP3受体与蛋白酶体偶联的蛋白。所采取的方法将是确定哪些蛋白质在IP3受体泛素化时与它们相关联，然后通过使用RNAi抑制IP3受体的表达来确定它们在IP3受体与蛋白酶体偶联中的作用。(3)触发UN受体泛素化事件的定义。所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动，以确定触发泛素化的IP3受体的因子和区域，然后检查这些区域可能的结构变化。

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.