Regulation of phospholipase C

描述(由申请人提供)：在过去的几年中，对肌醇脂质信号的复杂性的认识急剧增加。PtdIns(4，5)P2直接结合和调节许多蛋白质的活性，从这种次要的膜磷脂一步产生至少三个主要的第二信使。肌醇磷脂酶C同工酶整合了来自多个上游G蛋白调节因子的信号，是许多生长因子、激素、神经递质和其他刺激作用的中心，是组织严密但知之甚少的信号节点。我们实验室最近的工作增加了对Rac、Rho和异源三聚体G蛋白调节肌醇脂质信号的多种模式的认识。此外，我们的PLC-22和PLC-22在依赖GTP的与rac1的复合体中的结构提供了PLC同工酶的第一个机械快照，而不是十年前解决的PLC-4的原始结构。这一进展和最近在制造稳定的G1q和PLC-2同工酶复合体方面的成功使我们能够设计实验，从结构和力学上明确地理解G蛋白介导的PLC同工酶催化活性的调节。为了实现这一目标，我们提出了三个具体目标：具体目标1.阐明GTP结合的Rho与PLC-5结合并与RAS协同激活这一新的PLC同工酶的界面和机制。具体目的2.明确G1q与PLC-2同工酶羧基末端相互作用的结构基础，并阐明这种相互作用刺激PLC活性的机制。具体目的3.确定在含有磷脂酰肌醇的膜上PLC同工酶的界面激活过程中，催化结构域插入对活性部位的自抑制封闭被解除的机制。

DESCRIPTION (provided by applicant): Recognition of the complexities of inositol lipid signaling has increased dramatically in the last several years. PtdIns(4,5)P2 directly binds and regulates activity of many proteins, and at least three major second messengers are produced in a single step from this minor membrane phospholipid. The inositol lipid-specific phospholipase C isozymes integrate signals from multiple upstream G protein regulators, and act as highly organized but poorly understood signaling nodes central to the action of many growth factors, hormones, neurotransmitters, and other stimuli. Recent work from our laboratories has led to increased appreciation of multiple modes of regulation of inositol lipid signaling by Rac, Rho, and heterotrimeric G proteins. Moreover, our structures of PLC-22 and of PLC-22 in GTP-dependent complex with Rac1 provide the first mechanistic snapshots of PLC isozymes past the original structure of PLC-4 solved a decade ago. This progress and recent success in producing stable complexes of G1q and PLC-2 isozymes has placed us in position to design experiments that will provide understanding of G protein-mediated regulation of the catalytic activity of PLC isozymes in structurally and mechanistically unambiguous terms. We propose three specific aims to reach this goal: Specific Aim 1. To delineate the interface and mechanism through which GTP-bound Rho binds PLC-5 and cooperates with Ras in activation of this novel PLC isozyme. Specific Aim 2. To define the structural basis of interaction of G1q with the carboxy-terminus of PLC-2 isozymes, and to delineate the mechanism through which this interaction stimulates PLC activity. Specific Aim 3. To determine the mechanism whereby an auto-inhibiting occlusion of the active site by a catalytic domain insert is relieved during interfacial activation of PLC isozymes at phosphoinositide-containing membranes.