RECEPTOR REGULATION OF PHOSPHOINOSITIDE 3-KINASE

磷酸肌醇 3-激酶的受体调节

• 批准号: 2459512
• 负责人: ANDREW J MORRIS
• 金额: $ 17.06万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459512
• 关键词: G protein; adenosine triphosphate; cell free system; enzyme activity; enzyme substrate; high performance liquid chromatography; lipid metabolism; phosphatidylinositols; phosphorylation; phosphotransferases; protein tyrosine kinase; radiotracer; receptor coupling; second messengers; thin layer chromatography

In addition to their established role in the generation of second messengers by phospholipase C-catalysed hydrolysis, three of the inositol-containing phospholipids, and phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are substrates for a second receptor-regulated enzyme, phosphoinositide 3-kinase. This ATP-dependent enzyme catalyses the phosphorylation of the D-3 OH group of each of the above lipids and is activated by a range of growth factors and hormones that stimulate receptor tyrosine kinases and guanine nucleotide-dependent regulatory protein [G-protein]-linked receptors in a diverse variety of target cells. Accumulation of the products of phosphoinositide 3-kinase, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5- triphosphate is rapid in stimulated cells. It is probable that these 3- phosphorylated inositol lipids or some metabolite derived from them serves as an important signal in cellular responses to hormones and growth factors. Recent observations implicate phosphoinositide 3-kinase products as regulators of a calcium and phospholipid-dependent protein kinase, and modulators of protein secretion and trafficking. The long- term goal of the proposed research is to understand the cellular functions of receptor-stimulated formation of 3-phosphorylated inositol lipids. Two major questions will be addressed: of receptor-stimulated How do receptors, particularly those coupled to G-proteins, regulate phosphoinositide 3-kinase activity and what are the metabolic fates of the 3-phosphorylated inositol lipids when formed in stimulated cells. The specificity with which G-protein-coupled receptors stimulate the accumulation of 3-phosphorylated inositol lipids will be established. Cell lines expressing pathways for both tyrosine kinase receptor and G- protein-coupled receptor stimulation will be identified. The pathways by which these two classes of receptors regulate phosphoinositide 3- kinase will be compared in intact cells emphasizing the role of role of second messengers, protein kinases and G-proteins in this process. Using cell-free assays, the biochemical mechanisms by which G-protein-coupled receptors activate phosphoinositide 3-Kinase will be determined and the protein components of this system identified. The substrate-specificity of phosphoinositide 3-kinase and the metabolic interrelationships of the products of this enzyme and the other inositol-containing lipids will be examined in both intact and broken cell systems. The 3-phosphorylated inositol lipids will be prepared in radiolabeled and unlabeled forms. The metabolism of these lipids will be examined in cell and tissue homogenates. Successful completion of this program of research will greatly advance our understanding of the regulation and functions of phosphoinositide 3-kinase and its products, both of which promise to be pivotal components of an important signal transduction system that mediates cellular responses to stimulation by hormones and growth factors.

除了它们在第二代中的既定作用 通过磷脂酶C催化水解的使者，三个 含肌醇的磷脂和磷脂酰肌醇的磷脂， 磷脂酰肌醇4-磷酸盐和磷脂酰肌醇4,5-二磷酸盐 是第二受体调节的酶，磷酸肌醇的底物 3-激酶。 这种依赖ATP的酶催化了磷酸化的磷酸化 D-3 OH组的每个脂质，并被一系列范围激活 刺激受体酪氨酸激酶和激素的生长因子和激素 鸟嘌呤核苷酸依赖性调节蛋白[G蛋白]连接 各种靶细胞的受体。 积累 磷酸肌醇3-激酶的产物，磷脂酰肌醇3-磷酸的产物， 磷脂酰肌醇3,4-双磷酸和磷脂酰肌醇3,4,5- 三磷酸在刺激细胞中很快。 这些3-很可能 磷酸化的肌醇脂质或从中得出的一些代谢产物 在细胞对激素和激素的反应中充当重要信号 增长因素。 最近的观察结果暗示了磷酸肌醇3-激酶 产品作为钙和磷脂依赖性蛋白的调节剂 激酶以及蛋白质分泌和贩运的调节剂。 长期 拟议研究的一项目标是了解细胞 受体刺激的3-磷酸化肌醇形成的功能 脂质。 将解决两个主要问题：受体刺激 受体如何，特别是与G蛋白耦合的受体调节 磷酸肌醇3-激酶活性，什么是代谢命运 在刺激细胞中形成3磷酸化的肌醇脂质。 G蛋白偶联受体刺激的特异性 将建立3-磷酸化的肌醇脂质的积累。 表达酪氨酸激酶受体和G-的途径 将确定蛋白质偶联受体刺激。 路径 通过这两类受体调节磷酸肌醇3-- 在完整的细胞中，将比较激酶，以强调 在此过程中，第二个使者，蛋白激酶和G蛋白。 使用 无细胞测定，G蛋白偶联的生化机制 将确定受体激活磷酸肌醇3-激酶，并将 该系统的蛋白质成分确定。 底物特异性 磷酸肌醇3-激酶和代谢相互关系 该酶和其他含肌醇的脂质的产品将是 在完整和损坏的细胞系统中进行了检查。 3磷酸化 肌醇脂质将以放射性标记和未标记的形式制备。 这些脂质的代谢将在细胞和组织中检查 匀浆。 成功完成该研究计划将 大大提高了我们对调节和功能的理解 磷酸肌醇3-激酶及其产品，这两者都有承诺为 重要信号转导系统的关键组件 介导激素和生长对刺激的细胞反应 因素。