Activation of Phospholipase C beta by G Proteins

描述（由申请人提供）：异源三聚体G蛋白被膜受体激活，膜受体对激素、离子、光和神经递质等多种激动剂产生反应。活化的G蛋白反过来可以激活宿主的细胞内效应蛋白，其中之一是磷脂酶C-β（PLCb）。PLCb催化信号脂质磷脂酰肌醇4，5二磷酸水解，释放两个第二信使，导致细胞内钙增加和蛋白激酶C活化。G蛋白激活PLCb或其他效应物的机制尚不清楚，主要是由于缺乏关于PLCb - G蛋白复合物的结构信息。PLCb是一种多结构域酶，我们发现，γ亚基结合到这些结构域之一赋予激活的催化核心。在目标1中，我们将确定激活剂结合改变域间接触，从而增加催化的机制。我们还发现，根据激活条件，催化作用可以增加到不同的和不同的水平，从而打开了可以微调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中得到检验。通过G β γ亚基激活PLCb是长期的，但可以通过另一种蛋白质伴侣如G α（GDP）的存在改变PLCb-G β γ而显著降低。这一机制将在生物药理学和活细胞中进行研究，见目标3。在之前的资助期间，我们已经发现PLCb将结合并抑制非常强大的酶PLC δ，并且在信号传导期间释放β γ亚基将从复合物中置换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调节细胞中钙信号的能力。 G蛋白激活PLC β导致细胞内钙水平增加，进而激活多种蛋白质，导致细胞生长和分裂的变化。PLCb-G蛋白活化是诸如乙酰胆碱和大量药剂的试剂的关键细胞应答。该提案试图在分子水平上理解这种激活是如何发生的，相信在基本水平上理解这个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.