Pharmacological Study of G-proteins in Gene Regulation

描述（由申请人提供）：大量g蛋白偶联受体（gpcr）的激活导致细胞增殖，有助于卡波西氏肉瘤等疾病的进展。我们最近报道了gpcr激活核因子κ B (nf - κ B)，诱导大量负责细胞生长和存活的基因的表达。NF-KappaB的激活机制已经通过模型细胞因子如TNFalpha进行了广泛的表征，但对激活这一重要转录因子的g蛋白途径知之甚少。使用药物抑制剂和显性阴性DNA结构破坏g蛋白信号，我们发现g蛋白在激活NF-KappaB的能力上存在差异。虽然许多Galpha和β - γ亚基介导NF-KappaB的激活，但某些Galpha亚基也可以抑制NF-KappaB。在三个特定的目标中提出了实验，以确定g蛋白调节NFKappaB激活的机制。在Aim 1中，我们将研究G13激活NF-KappaB的信号通路。我们假设G13-p115RhoGEF-RhoA通路下游的信号效应物通过p65反激活在NF-KappaB激活中发挥关键作用。Aim 2的重点是β二聚体在PI-3激酶和Src蛋白酪氨酸激酶的差异激活中的作用，两者都导致NF-KappaB激活，但具有不同的1kappabeta - α动力学。我们建议确定的因素，决定激活这些效应的β - α。β -抑制素在β - γ介导的NF-KappaB激活中的作用也将被研究。在Aim 3中，我们将确定NF-KappaB活化是如何被g蛋白负调控的。一种可行的假设是，Galphai蛋白具有与gbetaggamma相反的功能，因为它们介导受促炎剂刺激的细胞中NF-KappaB活化的抑制。我们将研究Galphai2在抑制NF-KappaB中的可能作用。我们将研究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事件如何导致不同的细胞质和核信号传导和转录调节，并确定治疗干预的潜在位点。

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.