Regulation of phospholipase C

描述（由申请人提供）：在过去几年中，对肌醇脂质信号复杂性的认识急剧增加。PtdIns(4,5)P2直接结合并调节许多蛋白质的活性，并且至少有三个主要的第二信使是由这种次要的膜磷脂在一个步骤中产生的。肌醇脂质特异性磷脂酶C同工酶整合来自多个上游G蛋白调节因子的信号，并作为高度组织但鲜为人知的信号节点，对许多生长因子、激素、神经递质和其他刺激的作用至关重要。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的gtp依赖复合物与Rac1的结构提供了PLC同工酶的第一个机制快照，而不是十年前解决的PLC-4的原始结构。这一进展以及最近在生产G1q和PLC-2同工酶稳定复合物方面的成功，使我们能够设计实验，以结构和机制明确的方式理解G蛋白介导的PLC同工酶催化活性的调节。我们提出实现这一目标的三个具体目标：描述gtp结合的Rho结合PLC-5并与Ras合作激活这种新型PLC同工酶的界面和机制。具体目标2。定义G1q与PLC-2同工酶的羧基端相互作用的结构基础，并描述这种相互作用刺激PLC活性的机制。具体目标3。确定在含磷肌苷膜上PLC同工酶的界面激活过程中，催化结构域插入物对活性位点的自抑制阻塞得以缓解的机制。

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.