Activation of Phospholipase C beta by G Proteins

描述（由申请人提供）：异三聚体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有反应。活化的G蛋白反过来可以激活细胞内效应蛋白宿主，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的激活。由于缺乏PLCb - G蛋白复合物的结构信息，G蛋白激活PLCb或其他效应器的机制尚不清楚。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允许增加催化作用的域间接触的机制。我们还发现，根据活化条件的不同，催化作用可以增加到不同的水平，这开辟了PLCb活化可以微调以引发特定细胞反应的可能性。这个想法将在Aim 2中进行测试。Gbetagamma亚基对PLCb的激活是长期存在的，但可以通过另一种蛋白质伙伴如Galpha（GDP）的存在改变PLCb-Gbetagamma而显着降低。这一机制将在Aim 3的生物物理和活细胞中进行研究。在之前的资助期内，我们发现PLCb将结合并抑制非常强大的PLCdelta酶，并且在信号传导过程中释放的Gbetagamma亚基将从复合物中取代PLCb，从而激活PLCb并逆转PLCd抑制。在Aim 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。

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.