ACTIVATION OF PHOSPHOLIPASE C BETA BY G PROTEINS

异三聚体G蛋白被膜受体激活， 细胞外激素、神经递质和生长 因素然后G蛋白可以激活一个或多个效应分子， 一种未知的机制决定了信号 出行细胞含有许多受体、效应子和G蛋白， 互动.特定G-效应复合物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 从未检测过任何G蛋白和效应物。 我们的长期 目的是了解特定G蛋白 亚型激活特定的效应器。 在这两个不同的建议中， 将测试效应物激活的机制。此外，亲和力和 复合物的寿命，以及复合物的活化状态的持续时间。 效应器将被确定。我们将集中在效应器，磷脂酶 β 1（PLC β 1），主要由以下α亚基激活： G-q类蛋白质和PLC β 2，其主要由 β-γ亚基PLC β在信号传导中起着关键作用 这种途径的转导和破坏与疾病有关 细胞的状态。 我们将使用的主要实验技术是 实现这些目标的方法是荧光光谱法。 荧光方法 将使我们能够观察蛋白质的关联和构象变化， 真实的时间，并允许我们评估多重均衡。 此外，委员会认为， 荧光将使我们能够量化的能量和时间尺度 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-β 1和PLC β 2之间形成的复合物 使用稳态和停流方法，我们将确定是否 PLC β激活通过蛋白质向膜的募集而发生 表面的G蛋白，或是否激活是由于协会的G蛋白， 膜结合物种。然后我们将确定PLC β激活是否 通过构象变化而发生， 构象在G蛋白亚基解离后得以维持。 可能用于调节PLC β激活的次要因素将 例如膜表面的性质， 两种G蛋白亚基与不同PLC的同时缔合 同种型。

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.