ACTIVATION OF PHOSPHOLIPASE C BETA BY G PROTEINS

异源三聚体G蛋白被膜受体激活，膜受体对激素、离子和神经递质等多种激动剂产生反应。 G蛋白的α和β-γ亚基可以激活效应子。 其中之一，磷脂酶C-β（PLC-β），催化脂质磷脂酰肌醇4，5二磷酸（PIP 2）的水解，释放两种第二信使二酰基甘油和肌醇1，4，5三磷酸，这反过来又导致细胞内Ca 2+的增加和蛋白激酶C的活化。G蛋白亚基激活PLC-A的机制尚不清楚。最近，我们发现，如果我们用PLC δ的PH结构域替换PLC δ的普列克底物蛋白同源（PH）结构域（其不是G蛋白调节的），我们赋予G蛋白活化。 因此，G β-γ亚基对PLC β 2的激活通过PH结构域中的残基发生，PH结构域通过PLC的两个家族中保守的催化核心中的残基传递激活。 我们提出这导致活性位点的开放，允许中间体的更大释放和促进水解的增加的水含量。 PLC δ的催化核心的晶体结构是已知的，并且与PLC β高度同源。 在这里，我们将构建PLC β和PLC δ的突变体和嵌合蛋白，这将使我们能够确定G β-γ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些步骤被G β-γ亚基激活。 这将是第一项确定G蛋白激活界面效应物的分子机制的研究。尽管PLC-δ存在于所有哺乳动物细胞中，但其在细胞功能中的作用及其蛋白质调节剂尚不清楚。 我们意外地发现PLC-A与PLC-A强烈结合并抑制其高基础活性。 因此，从细胞刺激中释放G β-γ亚基可能会破坏PLC-β 2-PLC-δ 1的结合，导致细胞内Ca 2+的协同增加，并暗示PLC-δ是激素调节中的二级激活剂。 我们将通过体外和体内方法的独特组合来测试该模型。

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.