Pharmacological Study of G-proteins in Gene Regulation

描述（由申请人提供）：许多G蛋白偶联受体（GPCR）的激活导致细胞增殖，导致疾病进展，如卡波西肉瘤。我们最近报道了GPCR激活核因子κ B（NF-κ B），其诱导大量负责细胞生长和存活的基因的表达。NF-KappaB的激活机制已被广泛使用的模型细胞因子，如TNF α，但很少有人知道的G蛋白途径，激活这一重要的转录因子。使用药理学抑制剂和显性负性DNA结构破坏G蛋白信号传导，我们发现G蛋白激活NF-κ B的能力各不相同。虽然许多G α和G β γ亚基介导NF-κ B活化，但某些G α亚基也可以抑制NF-κ B。实验提出了3个具体的目的，以确定G蛋白调节NF κ B激活的机制。在目标1中，我们将研究G13用于NF-KappaB激活的信号通路。我们假设G13-p115 RhoGEF-RhoA通路下游的信号效应子通过p65反式激活在NF-κ B激活中起关键作用。目的2是集中在PI-3激酶和Src蛋白酪氨酸激酶的差异激活中的G β-γ二聚体，两者都导致NF-κ B激活，但具有不同的1 kappabeta α动力学。我们建议确定的因素，确定这些效应的Gbetaalpha的激活。还将检查β-抑制蛋白在γ-射线介导的NF-κ B活化中的作用。在目标3中，我们将确定NF-κ B激活如何受到G蛋白的负调控。一个工作假设是Galphai蛋白具有与G β γ相反的功能，因为它们介导用促炎剂刺激的细胞中NF-κ B活化的抑制。我们将研究Galpha 12在抑制NF-κ B中的可能作用。将检查Galkphai介导的Raf-1通路抑制的潜在联系。总的来说，这些研究有望揭示G蛋白介导的近端信号传导事件如何导致不同的细胞质和细胞核信号传导和转录调控，并确定潜在的治疗干预位点。

DESCRIPTION (provided by applicant): Activation of numerous G-protein-coupled receptors (GPCRs) results in cell proliferation that contributes to the progression of diseases such as Kaposi's sarcoma. We have recently reported that GPCRs activate nuclear factor kappa B (NF-kappaB), which induces the expression of a large number of genes responsible for cell growth and survival. The NF-KappaB activation mechanims have been extensively characterized using model cytokines such as TNFalpha, but little is known about the G-protein pathways that activate this important transcription factor. Using pharmacological inhibitors and dominant negative DNA constructs that disrupt G-protein signaling, we have found that G-proteins vary in their ability to activate NF-KappaB. While many Galpha and Gbetagamma subunits mediate NF-KappaB activation, certain Galpha subunits can also inhibit NF-KappaB. Experiments are proposed in 3 specific aims to determine the mechanisms by which G-proteins regulate NFKappaB activation. In Aim 1, we will investigate the signaling pathways utilized by G13 for NF-KappaB activation. We hypothesize that signaling effectors downstream of the G13-p115RhoGEF-RhoA pathway play a critical role in NF-KappaB activation through p65 transactivation. Aim 2 is focused on Gbetagamma dimers in the differential activation of PI-3 kinases and Src protein tyrosine kinases, both leading to NF-KappaB activation but with different 1kappabetaalpha kinetics. We propose to identify the factors that determine the activation of these effectors of Gbetaalpha. The role of beta-arrestins in Gbetagamma-mediated NF-KappaB activation will also be examined. In Aim 3, we will determine how NF-KappaB activation is negatively regulated by G-proteins. A working hypothesis is that Galphai proteins have the opposite function of Gbetagamma in that they mediate inhibition of NF-KappaB activation in cells stimulated with proinflammatory agents. We will investigate a possible role of Galphai2 in suppression of NF-KappaB. A potential link to Galkphai-mediated inhibition of Raf-1 pathway will be examined. Collectively, these studies are expected to reveal how G-protein mediated proximal signaling events lead to different cytoplasmic and nuclear signaling and transcriptional regulation, and to identify potential sites for therapeutic intervention.