Determinants of phospholipase C-Beta regulation

磷脂酶 C-Beta 调节的决定因素

• 批准号: 6383118
• 负责人: THERESA MARIE FILTZ
• 金额: $ 20.7万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6383118
• 关键词: G protein; Saccharomyces cerevisiae; biological signal transduction; calmodulin; cell line; cell membrane; chemical kinetics; enzyme induction /repression; genetic regulation; hydrolysis; immunoprecipitation; intermolecular interaction; matrix assisted laser desorption ionization; membrane lipids; molecular site; phosphatidylinositols; phospholipase C; phospholipids; phosphorylation; protein kinase; protein localization; protein purification; protein structure function; radiotracer; site directed mutagenesis; yeast two hybrid system

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.

肌醇磷脂水解的刺激是细胞内对跨膜受体被广泛的细胞外化学信号信使激活的初始反应。肌醇磷脂水解是由大多数胞外信号分子通过激活磷脂酶c - β (plc - β)酶的G蛋白亚基刺激的。肌醇磷脂水解的进一步调节（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通过激酶，脂质结合域和非g蛋白分子间相互作用准确，严格地描述plc - β 3同工酶调节。磷酸化介导的plc - β 3水解活性抑制动力学将在良好控制的体外测定中定量。将评估PLC- β 3分子内的脂质结合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，将鉴定plc - β 3与除G蛋白或激酶外的推定相互作用蛋白的关联，并在体内和体外定量这些分子间相互作用的功能。这些研究的目的是在分子水平上详细描述plc - β 3活性的多种调控手段，而不是已经被充分表征的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。详细的plc - β酶调控的功能图谱可能有助于设计新型治疗剂，选择性地干预plc - β介导的病理生理过程；与plc - β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、平滑肌肥大和增生、神经元激活和恶性细胞增殖等。