Activation of Phospholipase C beta by G Proteins

异源三聚体G蛋白被膜受体激活，这些受体对不同的激动剂有反应 包括激素、离子、光和神经递质。激活的G蛋白反过来可以激活宿主 细胞内效应蛋白，其中之一是磷脂酶C-β（PLCb）。PLCb催化 水解信号脂质磷脂酰肌醇4，5二磷酸以释放两个第二信使 导致细胞内钙离子增加和蛋白激酶C激活。有何机制 主要由于缺乏结构信息，G蛋白激活PLCb或其他效应物是未知的 关于PLCb - G蛋白复合物。PLCb是一种多结构域酶，我们发现， 亚基与这些结构域之一结合，赋予催化核心活化。在目标1中，我们将确定 激活剂结合改变域间接触的机制， 催化作用我们还发现，催化作用可以增加到不同的和不同的水平，这取决于 激活条件开启了PLCb激活可以被微调以引发 特殊的细胞反应。这一想法将在目标2中得到检验。通过G β γ亚单位激活PLC b 是长期存在的，但可以通过改变PLCb-β-γ通过存在显着减少 另一种蛋白质伴侣，如Galalpha（GDP）。这一机制将在生物药理学和 在Aim 3的活细胞中。在上一个资助期内，我们发现PLCb会约束和抑制 非常强大的酶PLC δ，并且在信号传导过程中释放的γ亚基将取代 从复合物中释放PLCb，允许PLCb的激活和PLCd抑制的逆转。在目标4中， 将更好地定义这两种PLC调节细胞中钙信号的能力。 G蛋白激活PLC β导致细胞内钙水平升高， 激活多种蛋白质，导致细胞生长和分裂的变化。PLCb - G蛋白激活是 对于诸如乙酰胆碱和大量药剂的药剂的关键细胞应答。这 该提案试图在分子水平上理解这种激活是如何发生的， 在基本水平上理解这一系统将允许设计更有效和更有针对性的药物。

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. InAim 1we 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 befine-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-Gbetagammathrough the presence of another protein partner such as Galpha(GDP). This mechanism will be investigated both biophysically and in living cells in Aim 3. Inthe 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 howthis activation occurs with the belief that understanding this system on a basic level will allow for the design of more effective and targeted drugs.