Activation of Phospholipase C beta by G Proteins

描述(申请人提供)：异三聚体G蛋白由膜受体激活，膜受体对一系列不同的激动剂做出反应，包括激素、离子、光和神经递质。激活的G蛋白又能激活一系列细胞内效应蛋白，其中之一是磷脂酶C-β(PLCB)。PLCB催化信号转导的磷脂酰肌醇4，5-二磷酸水解物释放两个第二信使，导致细胞内钙的增加和蛋白激酶C的激活。G蛋白激活PLCB或其他效应分子的机制尚不清楚，主要是由于缺乏PLCB-G蛋白复合体的结构信息。PLCB是一种多结构域酶，我们发现Gbetagamma亚基与这些结构域中的一个结合，从而激活催化核心。在目标1中，我们将确定激活剂结合改变结构域间接触从而增加催化的机制。我们还发现，根据激活条件的不同，催化作用可以增加到不同和不同的水平，从而打开了微调PLCB激活以引发特定细胞反应的可能性。这一想法将在目标2中得到验证。Gbetagamma亚单位对PLCB的激活是长期的，但通过改变PLCB-Gbetagamma，通过存在另一个蛋白质伙伴，如Galpha(GDP)，可以显著减少。这一机制将在Aim 3的生物物理和活细胞中进行研究。在之前的资助期间，我们发现PLCB将结合并抑制非常强大的酶PLCDelta，并且在信号传递过程中Gbetagamma亚单位的释放将取代PLCB，从而允许PLCB的激活和PLCd抑制的逆转。在目标4中，我们将更好地定义这两种PLC调节细胞内钙信号的能力。 G蛋白激活PLCbeta会导致细胞内钙离子水平的增加，进而激活多种蛋白质，导致细胞生长和分裂的变化。PLCB-G蛋白的激活是乙酰胆碱等药物和大量药物的关键细胞反应。这项建议试图在分子水平上了解这种激活是如何发生的，并相信在基础水平上了解这一系统将允许设计更有效和更有针对性的药物。

DESCRIPTION (provided by applicant): Heterotrimeric G proteins are activated by membrane receptors that respond to a diverse set of agonists ranging from hormones, ions, light and neurotransmitters. Activated G proteins in turn can activate a host of intracellular effector proteins, one of which is phospholipase C-beta (PLCb). PLCb catalyzes the hydrolysis of the signaling lipid phosphatidylinositol 4,5 bisphosphate to release two second messengers that cause an increase in intracellular calcium and activation of protein kinase C. The mechanism through which G proteins activate PLCb or other effectors is unknown mainly due to a lack of structural information about PLCb - G protein complexes. PLCb is a multidomain enzyme and we have found that Gbetagamma subunits bind to one of these domains confering activation to the catalytic core. In Aim 1 we will determine the mechanism through which activator binding changes interdomain contacts that allow for increased catalysis. We have also found that catalysis can be increased to different and distinct levels depending on the activation conditions opening up the possibility that activation of PLCb can be fine-tuned to elicit particular cellular responses. This idea will be tested in Aim 2. Activation of PLCb by Gbetagamma subunits is long-lived but can be significantly reduced by changing PLCb-Gbetagamma through the presence of another protein partner such as Galpha(GDP). This mechanism will be investigated both biophysically and in living cells in Aim 3. In the previous funding period, we have found that PLCb will bind to and inhibit the very robust enzyme PLCdelta, and that release of Gbetagamma subunits during signaling will displace PLCb from the complex allowing for both activation of PLCb and reversal of PLCd inhibition. In Aim 4 we will better define the ability of these two PLCs to regulate calcium signals in cells. Activation of PLCbeta by G proteins causes an increase in the cellular levels of calcium that in turn activates a variety of proteins leading changes in cell growth and division. PLCb - G protein activation is key cellular response for agents such as acetylcholine and a large number of pharmaceutical agents. This proposal seeks to understand on the molecular level how this activation occurs with the belief that understanding this system on a basic level will allow for the design of more effective and targeted drugs.