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。但是，当前的证据表明这些途径之间有明显的串扰。因此，G蛋白偶联受体的占用可以刺激酪氨酸激酶，鸟嘌呤核苷酸交换因子和调节途径的生长的活性。 G蛋白明显刺激磷脂酰肌醇3-激酶（PTDLNS 3-激酶）的p110-gamma同工型，从而导致磷脂酰肌醇（3,4,5）三磷酸盐的产生。 该脂质是一个重要信号，它激活磷脂酰肌醇依赖性蛋白激酶PDK-1，导致蛋白激酶B的激活和许多信号传导事件。该项目时期的重点是了解G蛋白α和β-伽马亚基激活PTDLNS 3激酶的机制。该目标将通过3个特定目标实现。 （AIM-1）确定哪种G蛋白α和β-Gamma亚基能够激活PTDLNS 3-激酶。 G蛋白家族的广泛多样化成员对效应子具有不同的活性。但是，尚不清楚该家族的哪个成员激活该酶。在此目标中，我们将确定定义组成的纯，重组G蛋白α亚基和β-gamma二聚体的能力，以激活PTDLNS 3激酶的纯化的P110-GAMMA同工型。 （AIM -2）了解激活PTDLNS 3激酶的β和伽马亚基中的域。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。在此目标中，我们将使用beta-gamma二聚体在β亚基中与突变和/或嵌合γ亚基结合使用的点突变来评估激活PTDLNS 3-激酶的β-伽马二聚体中的域。 （AIM-3）了解已知的调节机制如何影响β-gamma二聚体对PTDLNS 3激酶的活性。我们已经证明了两种影响β-gamma二聚体的新型调节机制。 （a）包含γ11亚基的二聚体无法刺激某些效应子，例如腺苷酸环化酶，并且； （b）含有γ12亚基的二聚体可以通过蛋白激酶C改变其活性来磷酸化。在此目标中，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。