Activation of Phospholipase C beta by G Proteins

异三聚体G蛋白被膜受体激活，膜受体对不同的激动剂做出反应 从荷尔蒙、离子、光和神经递质。被激活的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催化了 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 这会导致细胞内钙离子的增加和蛋白激酶C的激活。 由于缺乏结构信息，目前尚不清楚哪种G蛋白能激活PLCB或其他效应器 关于PLCB-G蛋白复合体。PLCB是一种多结构域酶，我们已经发现Gbetagamma 亚基结合到这些结构域中的一个，从而激活催化核心。在Aim 1wE中将确定 激活剂结合改变域间接触的机制允许增加 催化作用。我们还发现，催化作用可以增加到不同和不同的水平，这取决于 激活条件为PLCB的激活提供了一种可能性，即可以通过调节PLCB的激活来引发 特殊的细胞反应。这一想法将在AIM 2中得到验证。Gbetagamma亚基对PLCB的激活 是长期存在的，但可以通过改变PLCB-Gbetagam来显著减少 另一个蛋白质伙伴，如加尔法(GDP)。这一机制将从生物物理和 在AIM 3的活细胞中。在之前的资助期间，我们发现PLCB会结合并抑制 非常强健酶PLCDelta，在信号传递过程中Gbetagamma亚基的释放将取代 该复合体中的PLCB允许PLCB的激活和PLCd抑制的逆转。在Aim 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导致细胞内钙水平升高，进而 激活多种蛋白质，导致细胞生长和分裂的变化。PLCB-G蛋白激活是 乙酰胆碱等药物和大量药物的关键细胞反应。这 该提案试图在分子水平上理解这种激活是如何发生的，因为人们相信 在基础水平上了解这一系统将有助于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.