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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587635
• 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.

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。