Hormonal mechanisms of gene regulation

背景：胰岛素样生长因子-1 （IGF-1）启动的信号事件通常被描述为仅依赖于受体酪氨酸激酶激活。然而，一个独特的，依赖于G蛋白的途径也可能存在。虽然各种数据随后支持了这一初步观察，但解释G蛋白如何介导来自酪氨酸激酶受体的信号的机制尚未确定。相关发现：在之前的资助期内，我们确定了平滑肌细胞中IGF-1对G(i)的激活涉及G(i)与IGF-1受体的直接结合，并表明G- β / γ亚基激活介导了导致ERK1/2的途径。我们还发现了G(i)- α的几种新的效应物（ErbB2、EGFR、eIF-4E、组蛋白H2b）。后一项发现的一个独特之处在于确定了G(i)通过转激活机制偶联两种受体酪氨酸激酶IGF-1R和ErbB2的作用。假设：G(i)与IGF-1受体上的特定位点结合是ErbB2激活所必需的，ErbB2随后通过调节控制细胞骨架动力学的microrna的表达来促进细胞迁移。确定介导G(i)与IGF-1受体结合的结构因子。2 .探讨ErbB2在IGF-1信号转导、miRNA表达及细胞迁移中的作用。通过免疫印迹法生产用于监测单adp核糖基化的抗体。实验方法:1。gst标记的G(i)-alpha将作为下拉实验中重组活性IGF-1受体的诱饵。诱饵和受体之间的关系将通过免疫印迹法定量。G(i)- α与IGF-1受体的结合位点将通过重组IGF-1受体的系统突变来定位，最初通过删除10个氨基酸片段，然后在确定相互作用区域后进行单个点突变。然后将不能结合G(i)- α的代表性突变克隆到慢病毒载体中，并在IGF-1攻击的细胞中表达，以验证功能丧失。G(i)- α对配体依赖性和非配体依赖性ErbB2激活的作用将分别通过TAPI-2 （adam17特异性抑制剂）和细胞聚血素抑制剂SecinH3预处理，以及随后用IGF-1刺激来探索。ErbB2磷酸化将通过免疫印迹法监测。ErbB2在细胞迁移和miRNA表达中的作用将通过选择性抑制剂CP-724714进行评估。miRNA对erbb2依赖性迁移的贡献将通过选择性敲低miRNA来确定。我们设计了一种抗原来产生针对adp核糖化精氨酸的抗体。该试剂的制备将使我们能够使用Western blotting来筛选响应IGF-1处理的adp核糖化蛋白。意义：这项研究将阐明我们已经确定的新效应如何有助于典型酪氨酸激酶受体信号的转导。此外，我们还将提供酪氨酸激酶受体激活G蛋白的结构机制。目前已知的G(i)- α效应蛋白只有3种，它们在IGF-1信号转导中的作用尚不清楚。确定ErbB2激活的机制将有助于更好地理解它如何将G(i)- α与miRNA表达和细胞迁移结合起来，从而使我们能够为这种G蛋白如何促进RTK信号转导建立新的范例。最后，如果一种抗体能够被制造出来，我们将有一个工具，可以用来首次发现adp核糖基化是如何介导IGF-1信号的。

Background: Signalling events initiated by insulin-like growth factor-1 (IGF-1) have been typically described as being solely dependent upon receptor tyrosine kinase activation. However, a distinct, G protein-dependent pathway may also exist. While various data have subsequently supported this initial observation, a mechanism explaining how G proteins mediate signals deriving from tyrosine kinase receptors has not yet been identified. Relevant Findings: During the previous funding period, we established that activation of G(i) by IGF-1 in smooth muscle cells involves direct binding of G(i) to the IGF-1 receptor and showed that G-beta/gamma subunit activation mediates the pathway leading to ERK1/2. We also identified several novel effectors of G(i)-alpha (ErbB2, EGFR, eIF-4E, histone H2b). A unique feature of the latter finding is the identification of a role for G(i) in coupling two receptor tyrosine kinases, IGF-1R and ErbB2, via a transactivation mechanism.Hypothesis: Binding of G(i) to a specific site on the IGF-1 receptor is required for activation of ErbB2, which subsequently promotes cell migration by modulating expression of microRNAs that control cytoskeletal dynamics.Objectives:1. To determine the structural factors that mediate the binding of G(i) to the IGF-1 receptor.2. To explore the role of ErbB2 in IGF-1 signalling, miRNA expression and cell migration.3. To produce an antibody for monitoring mono-ADP-ribosylation by immunoblotting.Experimental Approach:1. GST-tagged G(i)-alpha will serve as bait for recombinant active IGF-1 receptor in a pull-down assay. The association between bait and receptor will be quantified by Western blotting. The binding site of G(i)-alpha with IGF-1 receptor will be mapped through systematic mutation of the recombinant IGF-1 receptor, initially by deletion of 10 amino acid sections and followed by individual point mutations once the region of interaction has been defined. A representative mutation that is incapable of binding G(i)-alpha will then be cloned in a lentivirus vector and expressed in cells challenged with IGF-1 to verify loss of function.2. The role of ligand-dependent and ligand-independent ErbB2 activation by G(i)-alpha will be explored via pretreatment with TAPI-2, an ADAM17-specific inhibitor, and the cytohesin inhibitor SecinH3, respectively, and subsequent stimulation with IGF-1. ErbB2 phosphorylation will be monitored by Western blotting. The role of ErbB2 in cell migration and miRNA expression will be assessed with a selective inhibitor, CP-724714. The contribution of miRNA to ErbB2-dependent migration will be identified by selective miRNA knockdown.3. We have designed an antigen for producing an antibody to ADP-ribosylated arginine. Preparation of this reagent will enable us to use Western blotting to screen for ADP-ribosylated proteins in response to IGF-1 treatment. Significance: This study will clarify how the new effectors we have identified contribute to the transduction of signals from canonical tyrosine kinase receptors. As well, we will provide a structural mechanism for the activation of a G protein by a tyrosine kinase receptor. At this point in time, only 3 effector proteins of G(i)-alpha are known, and their role in IGF-1 signal transduction is unknown. Identifying the mechanism for ErbB2 activation will provide a better understanding of how it couples G(i)-alpha to miRNA expression and cell migration, thus enabling us to establish a new paradigm for how this G protein contributes to RTK signal transduction. Finally, if an antibody can be produced, we would have a tool that could be used to discover, for the first time, how ADP-ribosylation mediates IGF-1 signalling.