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(GPCRs)的激活会导致细胞增殖，从而导致卡波西肉瘤等疾病的进展。我们最近报道了GPCRs激活核因子kappaB(NF-kappaB)，它诱导了大量与细胞生长和存活有关的基因的表达。核因子-kappaB的激活机制已经被广泛地用模型细胞因子如TNFpha来表征，但对激活这一重要转录因子的G蛋白途径知之甚少。使用药物抑制剂和干扰G-蛋白信号的显性负DNA结构，我们发现G-蛋白激活核因子-kappaB的能力不同。虽然许多Galpha和Gbetagamma亚基介导了NF-kappaB的激活，但某些Galpha亚基也可以抑制NF-kappaB。为了确定G蛋白调节NFKappaB激活的机制，提出了三个特定目的的实验。在目标1中，我们将研究G13激活核因子-kappaB的信号通路。我们假设G13-p115Rhogef-RhoA通路下游的信号效应分子通过p65反式激活在NF-kappaB激活中发挥关键作用。目的2重点研究Gbetagamma二聚体在PI-3和Src蛋白酪氨酸激酶的差异激活中的作用，这两种蛋白都导致了NF-kappaB的激活，但具有不同的1kappabetaα动力学。我们建议确定决定GbetaAlpha这些效应器激活的因素。还将研究β-拦阻素在Gbetagamma介导的NF-kappaB激活中的作用。在目标3中，我们将确定G蛋白如何负向调节NF-kappaB的激活。一个工作假说是Galphai蛋白具有与Gbetagamma相反的功能，因为它们介导了对促炎剂刺激的细胞中核因子-kappaB活性的抑制。我们将研究Galphai2在抑制核因子-kappaB中的可能作用。Galkphai介导的抑制Raf-1通路的潜在联系将被研究。总之，这些研究有望揭示G蛋白介导的近端信号事件如何导致不同的细胞质和核信号以及转录调控，并确定潜在的治疗干预部位。

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.