Beta gamma signaling from G protein linked receptors

来自 G 蛋白相关受体的 β-γ 信号传导

• 批准号: 6102232
• 负责人: JAMES Carlton GARRISON
• 金额: $ 15.64万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6102232
• 关键词: G protein; active sites; biological signal transduction; dimer; enzyme activity; isozymes; phosphatidylinositol 3 kinase; phosphatidylinositols; phospholipase C; protein protein interaction; protein structure function; protein tyrosine kinase; receptor coupling; receptor expression; recombinant proteins; tissue /cell culture

Historically, the signaling pathways used by G protein coupled receptors and receptor tyrosine kinases were considered very separate. However, current evidence indicates significant crosstalk between these pathways. Thus, occupation of G protein coupled receptors can stimulate the activity of tyrosine kinases, guanine nucleotide exchange factors and growth regulating pathways. G proteins markedly stimulate the p110-gamma isoform of phosphatidylinositol 3-kinase (Ptdlns 3-kinase) leading to production of phosphatidylinositol (3,4,5) trisphosphate. This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to activation of protein kinase B and a host of signaling events. The focus of this project period is to understand the mechanisms by which G protein alpha and beta-gamma subunits activate the Ptdlns 3-kinase. This goal will be approached via 3 Specific Aims. (Aim-1) To determine which G protein alpha and beta-gamma subunits are able to activate the Ptdlns 3-kinase. The broadly diverse members of the G protein family have differential activity on effectors. However, it is not known which members of the family activate this enzyme. In this aim, we will determine the ability of pure, recombinant G protein alpha subunits and beta-gamma dimers of defined composition to activate the purified p110-gamma isoform of Ptdlns 3-kinase. (Aim -2) To understand the domains in the beta and gamma subunits that activate Ptdlns 3-kinase. It is not known which regions of the Py dimer interact with the p110-gamma form of Ptdlns 3-kinase. In this aim we will use beta-gamma dimers with selected point mutations in the beta subunit in combination with mutated and/or chimeric gamma subunits to evaluate the domains in the beta- gamma dimer which activate the Ptdlns 3-kinase. (Aim-3) To understand how known regulatory mechanisms affect the activity of beta-gamma dimers on Ptdlns 3-kinase. We have demonstrated two novel regulatory mechanisms affecting the beta-gamma dimer. (a) Dimers containing the gamma11 subunit are not able to stimulate certain effectors such as adenylyl cyclase, and; (b) Dimers containing the gamma12 subunits can be phosphorylated by protein kinase C altering their activity. In this aim, we will examine how these and other regulatory mechanisms affect the ability of beta-gamma dimers to stimulate Ptdlns 3-kinase.

从历史上看，G 蛋白偶联受体和受体酪氨酸激酶使用的信号传导途径被认为是非常独立的。然而，目前的证据表明这些途径之间存在显着的串扰。因此，占领G蛋白偶联受体可以刺激酪氨酸激酶、鸟嘌呤核苷酸交换因子和生长调节途径的活性。 G 蛋白显着刺激磷脂酰肌醇 3-激酶 (Ptdlns 3-激酶) 的 p110-gamma 同工型，从而产生磷脂酰肌醇 (3,4,5) 三磷酸。 这种脂质是激活磷脂酰肌醇依赖性蛋白激酶 PDK-1 的重要信号，从而导致蛋白激酶 B 的激活和一系列信号事件。该项目期间的重点是了解 G 蛋白 alpha 和 beta-gamma 亚基激活 Ptdlns 3-激酶的机制。这一目标将通过 3 个具体目标来实现。 (Aim-1) 确定哪些 G 蛋白 α 和 β-gamma 亚基能够激活 Ptdlns 3-激酶。 G 蛋白家族的不同成员对效应子具有不同的活性。然而，尚不清楚该家族的哪些成员激活这种酶。为此，我们将确定确定组成的纯重组 G 蛋白 α 亚基和 β-γ 二聚体激活纯化的 Ptdlns 3-激酶 p110-γ 亚型的能力。 （目标 -2）了解激活 Ptdlns 3-激酶的 β 和 γ 亚基中的结构域。目前尚不清楚 Py 二聚体的哪些区域与 Ptdlns 3-激酶的 p110-gamma 形式相互作用。为此，我们将使用β亚基中具有选定点突变的β-γ二聚体与突变和/或嵌合γ亚基相结合来评估β-γ二聚体中激活Ptdlns 3-激酶的结构域。 (Aim-3) 了解已知的调控机制如何影响 Ptdlns 3-激酶上 β-γ 二聚体的活性。我们已经证明了两种影响β-γ二聚体的新颖调节机制。 (a) 含有 γ11 亚基的二聚体不能刺激某些效应子，例如腺苷酸环化酶； (b) 含有 γ12 亚基的二聚体可被蛋白激酶 C 磷酸化，从而改变其活性。为此，我们将研究这些和其他调节机制如何影响 β-γ 二聚体刺激 Ptdlns 3-激酶的能力。