Regulation of phospholipase C

描述（由申请人提供）：在过去几年中，对肌醇脂质信号传导的复杂性的认识显著增加。PtdIns（4，5）P2直接结合并调节许多蛋白质的活性，并且至少三种主要的第二信使在一个步骤中从这种次要的膜磷脂产生。肌醇脂质特异性磷脂酶C同工酶整合来自多个上游G蛋白调节剂的信号，并作为高度组织化但知之甚少的信号传导节点，对许多生长因子、激素、神经递质和其他刺激物的作用至关重要。我们实验室最近的工作使我们对Rac、Rho和异源三聚体G蛋白调节肌醇脂质信号传导的多种模式有了更多的认识。此外，我们的PLC-22和PLC-22在GTP依赖性复合物与Rac 1的结构提供了PLC同工酶过去的原始结构的PLC-4解决了十年前的第一个机械快照。这一进展和最近的成功，在生产稳定的复合物G1 q和PLC-2同工酶使我们能够设计实验，将提供G蛋白介导的调节PLC同工酶的催化活性的结构和机械上明确的条款的理解。我们提出了三个具体目标来实现这一目标：具体目标1。阐明GTP结合的Rho与PLC-5结合并与Ras协同激活这种新型PLC同工酶的界面和机制。具体目标2。确定G1 q与PLC-2同工酶羧基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。具体目标3。为了确定的机制，从而自动抑制闭塞的活性位点的催化结构域插入物被解除在界面激活的PLC同工酶在含磷酸肌醇的膜。

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.