ACTIVATION OF PHOSPHOLIPASE C BETA BY G PROTEINS

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、离子和神经递质。然后，G蛋白的α和β-伽马亚基可以激活效应器。其中，磷脂酶C-β(PLC-β)催化磷脂酰肌醇4，5-二磷酸(PIP2)的水解，释放两个第二信使--二酰甘油和1，4，5-三磷酸，进而导致细胞内钙离子的增加和蛋白激酶C的激活。最近，我们发现，如果将不受G蛋白调控的PLCDelta的Pleckstrin Homology(PH)结构域替换为PLCDelta的PH结构域，我们就可以激活G蛋白。因此，Gβ-γ亚基通过PH域的残基激活PLCbeta2，这些残基通过催化核心中保守的残基传递激活。我们认为这会导致活性中心的打开，从而允许更大的中间体释放和促进水解的水含量的增加。PLC Delta的催化核心的晶体结构是已知的，并且与PLCβ高度同源。在这里，我们将构建PLCβ和PLC Delta的突变和嵌合蛋白，这将使我们能够确定Gβ-伽马激活发生的分子途径。之后，我们将明确确定催化途径中的哪一步(S)被Gβ-伽马亚基激活。这将是第一次确定G蛋白激活界面效应器的分子机制的研究。尽管PLC-Delta在所有哺乳动物细胞中都有发现，但它在细胞功能中的作用以及它的蛋白质调节机制尚不清楚。我们意外地发现PLC-A与PLC-A结合强烈，并抑制其高基础活性。因此，从细胞刺激中释放Gβ-γ亚基可能会破坏PLC-β2-PLC-Delta1的结合，导致细胞内钙离子的协同增加，并暗示PLC-Delta是激素调节的二级激活剂。我们将通过一种独特的体外和体内相结合的方法来测试这个模型。

Heterotrimeric G proteins are activated by membrane receptors that respond to a diverse set of agonists ranging from hormones, ions and neurotransmitters. The alpha and beta-gamma subunits of G proteins can then activate effectors. One of these, phospholipase C-beta (PLC-beta), catalyzes the hydrolysis of the lipid phosphatidylinositol 4,5 bisphosphate (PIP2) to release the two second messengers diacylglycerol and inositol 1,4,5 trisphosphate which in turn result in an increase in intracellular Ca2+ and activation of protein kinase C. The mechanism through which G protein subunits activate PLC-A is unknown. Recently, we have found that if we replace the pleckstrin homology (PH) domain of PLCdelta, which is not G protein regulated, with the PH domain of PLCdelta, we confer G protein activation. Thus, activation of PLCbeta2 by G beta-gamma subunits occurs through residues in the PH domain, which transmit activation through residues in the catalytic core that are conserved among the two families of PLCs. We propose this results in an opening of the active site allowing for a greater release of intermediate and an increased water content which promotes hydrolysis. The crystal structure of the catalytic core of PLCdelta is known and is highly homologous to PLCbeta. Here, we will construct mutant and chimeric proteins of PLCbeta and PLCdelta that will allow us to determine the molecular' pathway through which G beta-gamma activation occurs. After, we will explicitly determine which step(s) in the catalytic pathway is activated by G beta-gamma subunits. This will be the first study to determine the molecular mechanism through which a G protein activates an interfacial effector. Even though PLC-delta is found in all mammalian cells, its role in cell function, and its protein regulators are unknown. We have unexpectedly found that PLC-A binds strongly to PLC-A and inhibits its high basal activity. Thus, release of G beta-gamma subunits from cell stimulation may disrupt PLC-beta2 - PLC-delta1 association causing a synergistic increase in intracellular Ca2+ and implicating PLC-delta as a secondary activator in hormonal regulation. We will test this model by a unique combination of in vitro and in vivo methods.