ACTIVATION OF PHOSPHOLIPASE C BETA BY G PROTEINS

异三聚体G蛋白被膜上的受体激活 细胞外激素、神经递质和生长的存在 各种因素。G蛋白可以通过以下方式激活一个或多个效应器分子 一种未知的机制，然后确定信号将通过的路径 旅行。细胞含有许多受体、效应器和G蛋白，可以 互动。特定G效应复合体的亲和力和持续时间是 预期是特定信号通路的基础，但这些参数 从未测量过任何G蛋白和效应器。我们的长期计划 目的是了解特定的G蛋白通过 子类型激活特定效应器。在这项提议中，有两个截然不同的 将测试效应器激活的机制。此外，亲和力和 络合物的寿命，以及激活状态的持续时间 效应器将被确定。我们将重点介绍磷脂酶的效应。 β1(PLCbeta1)，主要由α亚基激活 G-Q类蛋白质和PLCbeta2，它主要由 β-伽马亚基。PLCβ在信号转导中起着关键作用 这一通路的转导和中断与疾病有关 单元的状态。我们将使用的主要实验技术 实现这些目标的方法是荧光光谱学。荧光法 将使我们能够查看蛋白质的关联和构象变化 实时，并将使我们能够评估多个均衡。此外， 荧光将使我们能够量化能量和时间尺度 这些互动。在测量了它们的亲和力和寿命之后 G-α、G-β-γ与PLC-β1和PLC-β2形成的络合物 使用稳态和停流方法，我们将确定 PLCβ激活是通过将蛋白质募集到膜上来实现的 表面被G蛋白激活，或者激活是由于G蛋白的结合 膜结合型物种。然后我们将确定PLC Beta激活 通过构象改变发生，以及这是否被激活 G蛋白亚基解离后，构象得以维持。 可能用于调节PLCβ激活的次要因子随后将 被调查，如膜表面的性质和 两个G蛋白亚基与不同PLC的同时关联 异构体。

Heterotrimeric G proteins are activated by membrane receptors that respond to the presence of extracellular hormones, neurotransmitters and growth factors. G proteins can then activate one or more effectors molecules by an unknown mechanism which then determines the pathway the signal will travel. Cells contain many receptors, effectors and G proteins that can interact. The affinity and duration of a particular G-effector complex is expected to underlie the particular signal pathway, but these parameters have never been measured for any G protein and effector. Our long term goal is to understand the mechanism through which specific G protein subtypes activate specific effectors. In this proposal two distinct mechanisms of effector activation will be tested. Also, the affinity and lifetime of the complexes, and the duration of the activated state of the effector will be determined. We will focus on the effectors, phospholipase beta1 (PLCbeta1), which is primarily activated by the alpha subunits of the G-q class of proteins, and PLCbeta2, which is primarily activated by the beta-gamma subunits. PLCbeta plays a pivotal role in signal transduction and disruptions in this pathway is associated with disease states of the cell. The major experimental technique we will use to achieve these goals is fluorescence spectroscopy. Fluorescence methods will allow us to view protein associations and conformational changes in real time, and will allow us to assess multiple equilibria. Moreover, fluorescence will allow us to quantitate the energies and time scales of these interactions. After measuring the affinities and lifetimes of complexes formed between G-alpha, G-beta-gamma and PLC-beta1 and PLCbeta2 using steady state and stop-flow methods, we will determine whether PLCbeta activation occurs by recruitment of the protein to the membrane surface by G proteins, or whether activation is due to association of the membrane-bound species. We will then determine whether PLCbeta activation occurs through a conformational change, and whether this activated conformation is sustained upon dissociation of the G protein subunit. Secondary factors that may serve to regulate PLCbeta activation will then be investigated such as the nature of the membrane surface and simultaneous association of both G protein subunits to the different PLC isoforms.