G-protein Regulation of the Phosphatidyl Inositol (3,4,5) Trisphosphate Signal

磷脂酰肌醇 (3,4,5) 三磷酸信号的 G 蛋白调节

基本信息

批准号：
7570012
负责人：
JAMES Carlton GARRISON
金额：
$ 27.95万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2010-04-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7570012
关键词：
Adenosine Adhesions Adrenergic Receptor Alanine Amino Acids Blood Platelets Cell Line Cell Shape Cell membrane Cell physiology Cells Cyclic AMP Cyclic AMP-Dependent Protein Kinases Enzymes Event G-Protein-Coupled Receptors GTP-Binding Proteins Generations Goals Hematopoietic In Vitro Knockout Mice Laboratories Ligands Link Lipids Membrane Molecular Mutate Neutrophil Activation PH Domain PTEN gene Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Play Protein Isoforms Protein Kinase Protein Kinase C Proto-Oncogene Proteins c-akt Recombinant Proteins Regulation Relative (related person)Research Research Personnel Role Shapes Signal Pathway Signal Transduction Signaling Molecule Site Specificity Transmembrane Domain Work cell motility dimer eosinophil inositol-1,4,5-trisphosphate 5-phosphatase insight macrophage mast cell methionyl-leucyl-phenylalanine migration myo-inositol-1 (or 4)-monophosphatase neutrophil receptor receptor coupling research study response src Homology Region 2 Domain stoichiometry

项目摘要

G protein coupled receptors provide both positive and negative regulation of hematopoietic cells. In cells such as neutrophils, macrophages, mast cells and platelets, the p110y isoform of phosphatidylinositol (4,5) 3-kinase (Ptdlns 3-kinase) plays a major role in cell activation, shape changes and migration via generation of phosphatidylinositol (3,4,5) trisphosphate (PIPs). This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to phosphorylation of protein kinase B and a host of cell responses. The levels of PIP3 in the membrane of these cells are also tightly controlled by a SH2 domain containing inositol 5-phosphatase, SHIP, that is regulated by multiple mechanisms. Activation of G protein coupled receptors linked to Gi stimulates the p110y isoform of Ptdlns 3-kinase 40-60 fold by releasing specific py dimers. Activation of G protein coupled receptors linked to Gs raise cyclic AMP and can markedly inhibit the response of hematopoietic cells to stimulatory ligands. Our research has provided clear evidence of the specific isoforms of the Py dimer which activate the p110y isoform of Ptdlns 3 kinase. Recent experiments uncover the exciting result that both the p110y isoform of Ptdlns 3-kinase and SHIP can be phosphorylated by the cyclic AMP dependent protein kinase. The ability of the Py dimer to activate p101/p110y is inhibited by phosphorylation. Phosphorylation of SHIP activates the enzyme. These results provide a possible molecular explanation for the ability of cyclic AMP to inhibit the response of hematopoietic cells. The goal for this project is to determine the importance of these phosphorylation events in cell function. This goal will be approached via 2 Specific Aims. Aim-1a: To determine the effects of phosphorylation on the activity of Ptdlns 3-Kinase in vitro. Aim-1b: To understand how phosphorylation of Ptdlns 3-Kinase regulates the function of the enzyme in three cell lines. Aim-2a: To explore the effects of phosphorylation on the activity of SHIP in vitro. Aim-2b: To understand how phosphorylation of SHIP regulates its function in the intact cell. Completion of these Aims will provide considerable understanding of the molecular events regulating the levels of PIP3 in all cells and should reveal mechanisms by which G protein coupled receptors stimulate and inhibit the function of hematopoietic cells.

G 蛋白偶联受体对造血细胞提供正向和负向调节。在细胞中例如中性粒细胞、巨噬细胞、肥大细胞和血小板、磷脂酰肌醇的 p110y 亚型 (4,5) 3-激酶（Ptdlns 3-激酶）在细胞激活、形状变化和世代迁移中发挥着重要作用磷脂酰肌醇（3,4,5）三磷酸（PIP）。这种脂质是激活磷脂酰肌醇依赖性蛋白激酶 PDK-1，导致蛋白激酶 B 和 a 磷酸化细胞反应的宿主。这些细胞膜中 PIP3 的水平也受到 SH2 的严格控制。含有肌醇 5-磷酸酶 SHIP 的结构域，受多种机制调节。 G的激活与 Gi 连接的蛋白偶联受体通过释放刺激 Ptdlns 3-激酶的 p110y 亚型 40-60 倍特定的 py 二聚体。与 Gs 连接的 G 蛋白偶联受体的激活会提高环 AMP，并可显着抑制造血细胞对刺激配体的反应。我们的研究提供了明确的证据 Py 二聚体的特定同种型，可激活 Ptdlns 3 激酶的 p110y 同种型。最近的实验揭示了令人兴奋的结果：Ptdlns 3-激酶的 p110y 亚型和 SHIP 都可以被环 AMP 依赖性蛋白激酶磷酸化。 Py二聚体激活的能力 p101/p110y 被磷酸化抑制。 SHIP 的磷酸化会激活该酶。这些结果为环AMP抑制造血反应的能力提供了可能的分子解释细胞。该项目的目标是确定这些磷酸化事件在细胞功能中的重要性。这一目标将通过 2 个具体目标来实现。 Aim-1a：确定磷酸化对 Ptdlns 3-激酶的体外活性。 Aim-1b：了解 Ptdlns 3-激酶的磷酸化如何调节该酶在三种细胞系中的功能。 Aim-2a：探讨磷酸化对 SHIP 的体外活性。 Aim-2b：了解 SHIP 的磷酸化如何调节其在完整的细胞。完成这些目标将使人们对分子事件有深入的了解调节所有细胞中 PIP3 的水平，并应揭示 G 蛋白偶联受体的机制刺激和抑制造血细胞的功能。