Hormonal mechanisms of gene regulation

基因调控的激素机制

• 批准号: RGPIN-2014-04155
• 负责人: Zahradka, Peter
• 金额: $ 3.42万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=610864
• 关键词: Hormonal; mechanisms; gene; regulation

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.

工作背景：由胰岛素样生长因子-1（IGF-1）启动的信号传导事件通常被描述为仅依赖于受体酪氨酸激酶活化。然而，也可能存在不同的G蛋白依赖性途径。虽然随后的各种数据支持这一初步观察，解释G蛋白如何介导酪氨酸激酶受体衍生的信号的机制尚未确定。 相关发现：在之前的资助期间，我们确定了在平滑肌细胞中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ERK 1/2的途径。我们还鉴定了G（i）-α的几种新型效应物（ErbB 2、EGFR、eIF-4 E、组蛋白H2 b）。后一发现的独特特征是鉴定G（i）在通过反式激活机制偶联两种受体酪氨酸激酶IGF-1 R和ErbB 2中的作用。 假设：G（i）与IGF-1受体上的特定位点的结合是ErbB 2活化所必需的，ErbB 2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。 目的： 1.确定介导G（i）与IGF-1受体结合的结构因子。 2.探讨ErbB 2在IGF-1信号传导、miRNA表达和细胞迁移中的作用。 3.制备用于通过免疫印迹法监测单ADP核糖基化的抗体。 实验方法： 1. GST标记的G（i）-α将在下拉测定中用作重组活性IGF-1受体的诱饵。诱饵和受体之间的结合将通过蛋白质印迹法定量。G（i）-α与IGF-1受体的结合位点将通过重组IGF-1受体的系统突变进行定位，最初通过缺失10个氨基酸部分，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，并在用IGF-1攻击的细胞中表达，以验证功能丧失。 2.通过分别用TAPI-2（一种ADAM 17特异性抑制剂）和细胞粘连蛋白抑制剂SecinH 3预处理，随后用IGF-1刺激，将探索G（i）-α对配体依赖性和配体非依赖性ErbB 2活化的作用。将通过蛋白质印迹法监测ErbB 2磷酸化。ErbB 2在细胞迁移和miRNA表达中的作用将用选择性抑制剂CP-724714评估。将通过选择性miRNA敲低来鉴定miRNA对ErbB 2依赖性迁移的贡献。 3.我们设计了一种抗原，用于产生ADP-核糖基化精氨酸的抗体。该试剂的制备将使我们能够使用蛋白质印迹法来筛选响应IGF-1处理的ADP-核糖基化蛋白。 重要性：这项研究将阐明我们已经确定的新效应子如何促进经典酪氨酸激酶受体的信号转导。同时，我们将提供酪氨酸激酶受体激活G蛋白的结构机制。在这个时间点上，只有3个G（i）-α效应蛋白是已知的，它们在IGF-1信号转导中的作用是未知的。确定ErbB 2激活的机制将更好地了解G（i）-alpha如何与miRNA表达和细胞迁移偶联，从而使我们能够建立一个新的范式，了解G蛋白如何促进RTK信号转导。最后，如果可以产生抗体，我们将有一个工具，可以用来发现，第一次，ADP-核糖基化如何介导IGF-1信号。