INSP3 RECEPTOR UBIQUITINATION AND DOWN-REGULATION

近年来，我的研究重点一直是细胞表面受体激活后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（InsP 3）受体，蛋白质，形成通道的内质网（ER）膜，并响应InsP 3的结合，动员钙储存在ER内。 我的长期目标是（i）了解InsP 3受体在细胞内信号传导中的作用，（ii）确定调节InsP 3受体和其他信号蛋白的机制，以及（iii）建立InsP 3受体调节的生物学意义。自1991年以来，我一直在研究InsP 3受体下调，这是一种对细胞表面受体活化的新型适应性反应，可迅速降低细胞InsP 3受体含量，从而降低ER Ca 2+储存对InsP 3的敏感性。 最近，它已被证明，InsP 3受体下调介导的泛素/蛋白酶体途径，降解的许多细胞蛋白，目前被认为是一个治疗目标的关键途径。 InsP 3受体的泛素化是启动其降解的事件。 本发明的具体目的是（1）使用生物化学技术和InsP 3受体诱变来确定InsP 3受体中的泛素化位点，（2）使用生物化学技术和转染编码这些酶的cDNA来鉴定负责InsP 3受体泛素化的酶，（3）使用InsP 3受体诱变来确定加速InsP 3受体泛素化的信号传导事件，（4）研究InsP 3受体在大鼠脑内的泛素化和下调。这些目标的实现将定义导致InsP 3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;这种适应是细胞功能的许多生理修饰和对许多治疗和娱乐药物的耐受性的基础。 其次，它将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InsP 3受体泛素化和下调。第三，它将有助于绘制泛素/蛋白酶体通路，从而更好地了解旨在干扰这一过程的药物。

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.