DOWN-REGULATION OF INSP3 RECEPTORS

这是细胞的一个基本特征， 受刺激第二个使者和其他使者。 细胞内信号在细胞表面活化期间产生 受体不会保持恒定，但随着时间的推移趋于下降。在 近年来，我一直专注于细胞的适应性反应， 在受体（例如，毒蕈碱胆碱能受体）激活期间， 受体），刺激肌醇1，4，5-三磷酸（InsP 3）的形成。 1991年，我发现持续的毒蕈碱刺激SH-SY 5 Y 人神经母细胞瘤细胞减少了受体的细胞补体， 这抑制了InsP 3的主要细胞内功能 (Ca2+从内质网储存的移动）。后来我 表明这种下调涉及I型InsP 3的加速， 受体降解我的长期目标是定义机制， InsP 3受体下调的特异性，以充分表征其 对细胞功能的影响，并确定生理，病理或 在临床上，它是重要的。关于这一 应用程序，我的具体目标如下。首先，我将定义 I型InsP 3受体下调的机制。采用特定 抗体定量受体，我将使用蛋白酶抑制剂， 确定负责降解的活性，细胞器 干扰剂以限定下调的细胞内位点，和 表达突变的InsP 3受体的转染细胞，以确定区域 蛋白酶解的受体。最后为 定义蛋白水解途径的选择性，我将建立 其他内质网蛋白是否也下调。 其次，我将定义InsP 3受体的亚型特异性， 调控为此，我将提出针对II型和III型的抗血清 受体和I型受体的变体，并将研究 亚型受到下调。与此同时，我将使用这些 抗血清以确定不同亚型的相对丰度， 还将检查大鼠大脑的外植体，以评估 下调发生在更能代表体内 比细胞系。第三，我将说明 InsP 3受体下调对细胞功能的影响。为了测量这种 准确地说，我将用反义核酸抑制InsP 3受体的表达， 酸，并将监测对完整细胞中Ca 2+动员的影响。在 与这些研究平行，我将使用反义核酸来定义 II型和III型受体的细胞内作用。的影响 受体表达的抑制，以及 不同的β 3受体亚型的功能和相对丰度， 将揭示InsP 3受体下调如何改变细胞功能- 由持续的细胞表面受体激活引起的调节。 总之，这些研究将提供一个全面的图片， InsP 3受体下调的机制、特异性和意义。

It is a basic characteristic of cells that they adapt when persistently stimulated. Thus, the extent to which second messengers and other intracellular signals are generated during activation of cell surface receptors does not remain constant, but with time tends to decline. In recent years, I have focussed on the adaptive responses that cells employ during activation of receptors (for example, muscarinic cholinergic receptors) that stimulate inositol 1,4,5-trisphosphate (InsP3) formation. In 1991, I discovered that persistent muscarinic stimulation of SH-SY5Y human neuroblastoma cells reduced the cellular complement of receptors for InsP3 and that this suppressed the primary intracellular function of InsP3 (Ca2+ mobilization from endoplasmic reticular stores). Subsequently, I showed that this down-regulation involved an acceleration of type l InsP3 receptor degradation. My long term goals are to define the mechanism and specificity of InsP3 receptor downregulation, to fully characterize its effects on cell function and to identify physiological, pathological or clinical situations in which it is significant. With regard to this application, my specific aims are as follows. Firstly, I will define the mechanism of type I InsP3 receptor down-regulation. Employing a specific antibody to quantitate receptors, I will use protease inhibitors to identify the activity responsible for the degradation, organelle perturbants to define the intracellular site of down-regulation, and transfected cells expressing mutated InsP3 receptors to define the regions of the receptor that are subject to proteolytic cleavage. Finally, to define the selectivity of the proteolytic pathway, I will establish whether other endoplasmic reticulum proteins are also down-regulated. Secondly, I will define the subtype specificity of InsP3 receptor down- regulation. To this end, I will raise antisera specific to type II and III receptors and to variants of the type I receptor and will examine which subtypes are subject to down-regulation. In parallel, I will use these antisera to determine the relative abundance of the different subtypes and will also examine explants of rat brain to assess the extent to which down-regulation occurs in systems more representative of the in vivo situation than cell lines. Thirdly, I will define the consequences of InsP3 receptor down-regulation on cell function. To measure this precisely, I will inhibit InsP3 receptor expression with antisense nucleic acids and will monitor effects on Ca2+ mobilization in intact cells. In parallel with these studies, I will use antisense nucleic acids to define the intracellular roles of type II and III receptors. The effects of inhibition of receptor expression, together with knowledge of the functions and relative abundance of the different Insp3 receptor subtypes, will reveal how cell function is altered by the InsP3 receptor down- regulation that results from persistent cell surface receptor activation. In summary, these studies will provide a comprehensive picture of the mechanism, specificity and significance of InsP3 receptor down-regulation.