DOWN-REGULATION OF INSP3 RECEPTORS

这是细胞的一个基本特征，当它们持久地适应 受到刺激。因此，第二个信使和其他人 细胞内信号是在细胞表面激活过程中产生的 受体并不是保持不变，而是随着时间的推移而趋于下降。在……里面 近年来，我一直专注于细胞利用的适应性反应 在受体激活期间(例如，M胆碱能 受体)刺激肌醇1，4，5-三磷酸(InsP3)的形成。 1991年，我发现对SH-SY5Y的持续性毒碱刺激 人神经母细胞瘤细胞减少受体的细胞补体 InsP3以及这抑制了InsP3的主要细胞内功能 (从内质网状库中动员钙离子)。随后，我 显示这种下调涉及L在P3型的加速 受体退化。我的长期目标是定义机制和 InsP3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 在临床情况下，它是重要的。对此， 申请，我的具体目标如下。首先，我将定义 I型InsP3受体下调的机制。使用特定的 为了量化受体的抗体，我将使用蛋白酶抑制剂来 确定负责降解的活动，细胞器 微扰来定义下调的细胞内位置，以及 表达突变InsP3受体的转基因细胞确定区域 容易被蛋白质分解的受体的。最后，为了 定义蛋白质分解途径的选择性，我将建立 其他内质网蛋白是否也下调。 其次，我将定义InsP3受体down的亚型特异性- 监管。为此，我将提出针对III型和III型的抗血清。 受体和I型受体的变体，并将检查哪些 亚类受到下调的监管。同时，我将使用这些 抗血清以确定不同亚型的相对丰度 还将检查老鼠大脑的外植体，以评估在多大程度上 下调发生在更具体内代表性的系统中 情况比细胞系要好。第三，我将定义 InsP3受体下调细胞功能。来衡量这个 准确地说，我会用反义核酸抑制InsP3受体的表达 并将监测对完整细胞内钙离子动员的影响。在……里面 在这些研究的同时，我将使用反义核酸来定义 III型和III型受体的细胞内作用。的影响 受体表达的抑制，以及对 不同Insp3受体亚型的功能和相对丰度， 将揭示InsP3受体下调是如何改变细胞功能的- 由于持续的细胞表面受体激活而产生的调节。 总括而言，这些研究将提供有关 InsP3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.