STRUCTURE AND FUNCTION OF PHOSPHOLIPASE C ENZYMES

磷脂酰肌醇特异性磷脂酶C的调控 激素的活动控制着两个第二信使IP3的进化 和二酰甘油，以及它们对细胞内游离的影响 钙通道蛋白2与Gq蛋白激酶C、G蛋白的活性 亚家族被认为是PLCbeta1活性的调节因子。我们 已经将这一规定扩展到另外两种同种类型的PLC Beta，即PLC Beta2 和PLCbeta3。PIPLC的所有三种同种类型都可以激活到不同的 由α-Q和β-伽马亚单位计算的范围。 此应用程序旨在将这些研究扩展到三个主要方面 方向。首先，我们建议确定结构性决定因素 G对PLCβ酶的调节很重要 蛋白质和一般作用。这将通过表达式和 杆状病毒表达系统对突变蛋白的鉴定。 其次，我们计划寻找其他对α-Q有反应的PIPLC酶 和/或β-伽马亚基。这将包括用原油进行实验。 从组织和细胞中制备以及表征 表达的PLC Delta异构体。最后，初步证据表明， 纯化的PLCbeta3含有抑制亚基。提出了计划 以验证这一观察结果，并对抑制蛋白进行表征。 我们还建议确定是否存在其他假定的抑制亚基。 对于PLCbeta1和PLCbeta2。 这项工作将增加我们对G蛋白依赖性的理解 对细胞内游离钙和蛋白质磷酸化的调节作用 荷尔蒙。更好地理解PIPLC的调节机制 将有助于开发更好的工具来操纵他们的 功能。PLCβ特异性抑制亚基的鉴定 同种异型将建立一个潜在的强大的工具来解剖 这些单独的酶在细胞内调节中的作用。

The regulation of phosphatidylinositol-specific phospholipase C (PIPLC) activity by hormones controls the evolution of two second messengers, IP3 and diacylglycerol, and their subsequent effects on intracellular free Ca2+ and the activity of protein kinase C. G proteins of the Gq subfamily have been identified as mediators of PLCbeta1 activity. We have extended this regulation to two other isotypes of PLCbeta, PLCbeta2 and PLCbeta3. All three isotypes of PIPLC can be activated to different extents by both alpha-q and beta-gamma subunits. This application is designed to extend these studies in three major directions. First, we propose to determine the structural determinants of the PLCbeta enzymes that are important for their .regulation by G proteins and action in general. This will be done through expression and characterization of mutant proteins with a Baculovirus expression system. Second, we plan to search for other PIPLC enzymes that respond to alpha-q and/or beta-gamma subunits. This will include experiments with crude preparations from tissues and cells as well as characterization of expressed PLCdelta isoforms. Finally, initial evidence suggests that the purified PLCbeta3 contains an inhibitory subunit. Plans are presented to verify this observation and to characterize the inhibitory protein. We also propose to determine if other putative inhibitory subunits exist for PLCbeta1 and PLCbeta2. This work will increase our understanding of G protein dependent regulation of intracellular free Ca2+ and protein phosphorylation by hormones. A better understanding of the mechanisms of PIPLC regulation will help in the development of better tools to manipulate their function. The identification of specific inhibitory subunits for PLCbeta isotypes would establish a potentially powerful tool for dissecting the roles of these individual enzymes in intracellular regulation.