Determinants of phospholipase C-Beta regulation

刺激肌醇磷脂的水解是细胞内对跨膜受体激活的初始反应，它是由一系列细胞外化学信号信使激活的。大多数细胞外信号分子通过激活磷脂酶C-β(PLC-β)的G蛋白亚基来刺激肌醇磷脂的水解。肌醇磷脂水解酶的进一步调节(如抑制)可能由其他因素介导，包括共价修饰和接近膜结合底物。该项目的长期目标是准确、严格地描述通过激动酶、脂结合结构域和非G蛋白分子间相互作用调节PLC-Beta3同工酶。磷酸化介导的抑制PLC-Beta3水解酶活性的动力学将在良好控制的体外检测中定量。将评估PLC-β3分子内假定的脂结合结构域的磷脂和膜结合亲和力，以进一步将结构与PLC-β活性的功能和调节联系起来。此外，将确定PLC-beta3与G蛋白或激酶以外的假定相互作用蛋白的联系，并对这些分子间相互作用的功能进行体内和体外的定量测定。这些研究的目的是在分子水平上详细说明PLC-β3活性的多种调控手段，而不是已经很好地描述的G蛋白激活途径，这将有助于更好地理解信号转导的基本机制。PLC-beta酶调控的详细功能图可能有助于设计选择性干预PLC-beta介导的病理生理过程的新型治疗剂；与PLC-beta激活相关的激素和神经递质反应包括平滑肌收缩、血小板聚集、激素分泌、平滑肌肥大和增殖、神经元激活和恶性细胞增殖等。

Stimulation of inositol phospholipid hydrolysis is the initial intracellular response to transmembrane receptor activation by a wide array of extracellular chemical signaling messengers. Inositol phospholipid hydrolysis is stimulated by most extracellular signaling molecules through G protein subunits which activate phospholipase C-beta (PLC-beta) enzymes. Further regulation (e.g. inhibition) of inositol phospholipid hydrolysis may be mediated by alternative factors including covalent modification and proximity to membrane bound substrate. This project's long-term goal is an accurate, rigorous description of PLC-beta3 isoenzyme regulation by kinases, lipid binding domains, and non-G protein intermolecular interactions. The kinetics of phosphorylation-mediated inhibition of PLC-beta3 hydrolytic activity will be quantitated in well-controlled in vitro assays. Phospholipid and membrane binding affinities of putative lipid binding domains within the PLC-beta3 molecule will be assessed to further associate structure with function and regulation of PLC- beta activity. Additionally, the association of PLC-beta3 with putative interacting proteins, other than G proteins or kinases, will be identified and the function of these intermolecular interactions quantitated both in vivo and in vitro. These studies, which aim to detail at a molecular level multiple means of regulation of PLC-beta3 activity beyond the well-characterized G protein activation pathway, will contribute to a greater understanding of the basic mechanisms of signal transduction. A detailed, functional map of PLC-beta enzyme regulation may contribute to the design of novel therapeutic agents that intervene selectively into pathophysiological PLC-beta-mediated processes; hormone and neurotransmitter responses associated with PLC-beta activation include, among many others, smooth muscle contraction, platelet aggregation, hormone secretion, smooth muscle hypertrophy and hyperplasia, neuronal activation, and malignant cell proliferation.