Beta gamma signaling from G protein linked receptors

历史上，G蛋白偶联受体和受体酪氨酸激酶使用的信号通路被认为是非常独立的。然而，目前的证据表明，这些通路之间存在显著的串扰。因此，G蛋白偶联受体的占据可以刺激酪氨酸激酶、鸟嘌呤核苷酸交换因子和生长调节通路的活性。G蛋白显著刺激p110-γ亚型磷脂酰肌醇3-激酶(Ptdlns 3-Kinase)，导致磷脂酰肌醇(3，4，5)三磷酸的产生。这种脂质是一个重要的信号，激活磷脂酰肌醇依赖的蛋白激酶PDK-1，导致蛋白激酶B的激活和一系列信号事件。本项目的重点是了解G蛋白α和β-伽马亚基激活Ptdlns3-激酶的机制。这一目标将通过三个具体目标来实现。(AIM-1)确定哪些G蛋白α和β-伽马亚基能够激活Ptdlns 3-激酶。G蛋白家族中广泛不同的成员对效应器有不同的活性。然而，目前还不知道该家族的哪些成员激活了这种酶。在这一目标中，我们将确定纯的、重组的G蛋白α亚基和确定组成的β-伽马二聚体激活纯化的Ptdlns 3-激酶的p110-伽马亚型的能力。(目的-2)了解激活Ptdlns 3-激酶的β和伽马亚基中的结构域。目前还不知道Py二聚体的哪些区域与Ptdlns 3-Kinase的p110-Gamma形式相互作用。为了达到这个目的，我们将结合突变和/或嵌合的伽马亚基来评估激活Ptdlns3-激酶的β-伽马二聚体中的结构域。(目的-3)了解已知的调节机制如何影响Ptdlns 3-K上的β-伽马二聚体的活性。我们已经证明了影响β-伽马二聚体的两种新的调控机制。(A)含有Gamma11亚基的二聚体不能刺激某些效应器，如腺酰环化酶；以及；(B)含有Gamma12亚基的二聚体可以被蛋白激酶C磷酸化，从而改变其活性。在这个目标中，我们将研究这些和其他调节机制如何影响β-伽马二聚体刺激Ptdlns 3-激酶的能力。

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.