STRUCTURE AND FUNCTION OF PHOSPHOLIPASE C ENZYMES

磷脂酰肌醇特异性磷脂酶C（PIPLC）的调控 激素的活性控制着两个第二信使IP 3的进化 和甘油二酯，以及它们对细胞内游离 Ca ~（2+）和蛋白激酶C活性。 Gq蛋白 亚家族已被鉴定为PLC β 1活性的介导物。 我们 我已经将这种调节扩展到PLC β的另外两种同种型，PLC β 2 PLC β 3 PIPLC的所有三种同种型都可以被激活以不同的方式表达。 由α-q和β-γ亚基延伸。 此应用程序旨在将这些研究扩展到三个主要领域 方向 首先，我们建议确定结构决定因素 的PLC β酶，这是重要的，他们的调节G 蛋白质和一般作用。 这将通过表达和 用杆状病毒表达系统表征突变蛋白。 其次，我们计划寻找其他对α-q有反应的PIPLC酶， 和/或β-γ亚基。 这将包括原油的实验 组织和细胞的制备以及表征 表达PLC δ同种型。 最后，初步证据表明， 纯化的PLC β 3含有抑制亚基。 提出了计划 以验证该观察结果并表征抑制性蛋白。 我们还建议确定是否存在其他假定的抑制亚基 对于PLC β 1和PLC β 2。 这项工作将增加我们对G蛋白依赖性 调节细胞内游离Ca 2+和蛋白质磷酸化 荷尔蒙 更好地理解PIPLC调节机制 将有助于开发更好的工具来操纵他们的 功能 PLC β特异性抑制亚基的鉴定 同种型将建立一个潜在的强大工具， 这些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.