GPCR Signaling & Vascular Wall Remodeling

GPCR 信号转导

• 批准号: 8092058
• 负责人: GADIPARTHI N RAO
• 金额: $ 43.89万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8092058
• 关键词: Address; Agonist; Allografting; Angioplasty; Applications Grants; Arteries; Atherosclerosis; Attention; Balloon Angioplasty; Binding Proteins; Biological Assay; Blood Coagulation Factor; Blood Vessels; Blood coagulation; Bone Marrow Transplantation; Cell Proliferation; Cloning; Co-Immunoprecipitations; Complex; Development; Disease; Dominant-Negative Mutation; Drug Controls; EGF gene; Epidermal Growth Factor; Epidermal Growth Factor Receptor; F-Actin; F2R gene; Fluorescence; Fluorescence Microscopy; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GRB2 gene; GTP-Binding Proteins; Goals; Guanosine Triphosphate; Heparin Binding; Heparin Binding Growth Factor; Human; Immunofluorescence Immunologic; In Vitro; Injury; Knockout Mice; Knowledge; Lesion; Ligands; MALDI-TOF Mass Spectrometry; Measurement; Mechanics; Mediating; Mediation; Mitogens; Molecular; PAR-1 Receptor; PAR-2 Receptor; Peptide Hydrolases; Peripheral Vascular Diseases; Phosphotransferases; Play; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteinase-Activated Receptors; Receptor Activation; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research Proposals; Role; Serine; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle Myocytes; Stimulation of Cell Proliferation; Stress Fibers; Testing; Therapeutic Agents; Threonine; Thrombin; Transactivation; Transgenic Mice; Veins; base; cell motility; cell type; disorder control; in vitro Assay; in vivo; inhibitor/antagonist; macrophage; migration; neointima formation; novel; protease-activated receptor 3; protease-activated receptor 4; protein protein interaction; receptor; research study; response; restenosis; rho GTP-Binding Proteins; scaffold; tool; vascular smooth muscle cell migration; Address; Agonist; Allografting; Angioplasty; Applications Grants; Arteries; Atherosclerosis; Attention; Balloon Angioplasty; Binding Proteins; Biological Assay; Blood Coagulation Factor; Blood Vessels; Blood coagulation; Bone Marrow Transplantation; Cell Proliferation; Cloning; Co-Immunoprecipitations; Complex; Development; Disease; Dominant-Negative Mutation; Drug Controls; EGF gene; Epidermal Growth Factor; Epidermal Growth Factor Receptor; F-Actin; F2R gene; Fluorescence; Fluorescence Microscopy; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GRB2 gene; GTP-Binding Proteins; Goals; Guanosine Triphosphate; Heparin Binding; Heparin Binding Growth Factor; Human; Immunofluorescence Immunologic; In Vitro; Injury; Knockout Mice; Knowledge; Lesion; Ligands; MALDI-TOF Mass Spectrometry; Measurement; Mechanics; Mediating; Mediation; Mitogens; Molecular; PAR-1 Receptor; PAR-2 Receptor; Peptide Hydrolases; Peripheral Vascular Diseases; Phosphotransferases; Play; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteinase-Activated Receptors; Receptor Activation; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research Proposals; Role; Serine; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle Myocytes; Stimulation of Cell Proliferation; Stress Fibers; Testing; Therapeutic Agents; Threonine; Thrombin; Transactivation; Transgenic Mice; Veins; base; cell motility; cell type; disorder control; in vitro Assay; in vivo; inhibitor/antagonist; macrophage; migration; neointima formation; novel; protease-activated receptor 3; protease-activated receptor 4; protein protein interaction; receptor; research study; response; restenosis; rho GTP-Binding Proteins; scaffold; tool; vascular smooth muscle cell migration

DESCRIPTION (provided by applicant): Thrombin, a serine/threonine protease, besides its role in blood coagulation, plays a significant role as a mitogen and motogen to many cell types, in particular to vascular smooth muscle cells (VSMCs). Thrombin mediates its effects via protease-activated receptors (PARs), namely PAR-1, PAR-2, PAR-3 and PAR-4. Downstream to PARs, it requires the need for the participation of G proteins, notably Gq/11 or G12/13 in the regulation of cell migration and proliferation. In addition, thrombin possesses the capacity to transactivate receptor tyrosine kinases (RTKs), among which epidermal growth factor receptor (EGFR) gained more attention. While protease-dependent shedding of heparin-binding growth factors, in this case, heparin-binding epidermal growth factor (HB-EGF), appears to be accountable for thrombin transactivation of EGFR, there appears to be a gap in our understanding of how this EGFR transactivation influences thrombin-induced VSMC mitogenesis and motogenesis. The major question is, is transactivation of EGFR sufficient in the stimulation of mitogenic and/or motogenic signal flows downstream to the receptor? Towards elucidating these signal flows, we discovered that thrombin activates GRB2-associated binding protein 1 (Gab1) and Src homology 2- containing protein tyrosine phosphatase (Shp2), whose stimulation is otherwise expected in response to EGFR activation by its true ligand, EGF, and the stimulation of this multifunctional signaling complex requires EGFR tyrosine kinase activity. What is more exciting is that Gab1-Shp2 activation is required for thrombin-induced Rho GTPase stimulation and F-actin stress fiber formation. Based on these novel observations, we propose to test the following specific aims with a goal to elucidate the G protein-coupled receptor (GPCR) signal flows that are upstream and downstream to EGFR transactivation in human aortic smooth muscle cells (HASMCs) and test their strength in the mediation of F-actin stress fiber formation, migration and proliferation of these cells in response to thrombin in vitro and vascular wall remodeling after angioplasty in vivo. The specific aims that will be addressed in this research proposal are as follows: 1. Thrombin-induced HASMC F-actin stress fiber formation, migration, proliferation and neointima formation require Gab1 activation. 2. Thrombin activates Rho GTPases via recruitment of RhoGEFs by Gab1 and RhoGEF-dependent RhoA, Rac1 and Cdc42 activation mediate HASMC F-actin stress fiber formation, migration, proliferation and neointima formation. 3. Gab1 targets PAK1 in mediating thrombin-induced HASMC F-actin stress fiber formation, migration, proliferation and neointima formation. Briefly, the results of the proposed experiments will fill the gap in our understanding of how GPCR signaling via crosstalk with RTK signaling and targeting a scaffold adaptor molecule, Gab1, leading to RhoGEF-mediated RhoA-Rac1/Cdc42-PAK1 activation plays a role in vascular wall remodeling following injury. Such comprehensive knowledge on the pathobiology of vascular wall diseases could become a valuable tool in the development of drugs for the control of these vascular lesions. PUBLIC HEALTH RELEVANCE: Vascular smooth muscle cell (VSMC) migration and proliferation play an important role in peripheral vascular diseases such as atherosclerosis and restenosis following angioplasty or vein grafting. Elucidating the molecular mechanisms underlying VSMC migration and proliferation is crucial in the development of therapeutic agents to control the disease process of these vascular lesions. In this regard, the present grant proposal seeks to study the mechanisms by which thrombin, a G protein-coupled receptor agonist and a clotting factor that is produced at the site of vascular injury could transactivate receptor tyrosine kinase signaling in the stimulation of both VSMC migration and proliferation and ignite the disease process.

描述（由申请人提供）：凝血酶，一种丝氨酸/苏氨酸蛋白酶，除了其在血液凝结中的作用外，还具有许多细胞类型的丝裂原和Motogen的重要作用，尤其是血管平滑肌细胞（VSMC）。凝血酶通过蛋白酶激活的受体（PARS），即PAR-1，PAR-2，PAR-3和PAR-4介导其作用。下游到PAR，它需要需要G蛋白的参与，尤其是GQ/11或G12/13在细胞迁移和增殖的调节中。此外，凝血酶具有反式激活受体酪氨酸激酶（RTK）的能力，其中表皮生长因子受体（EGFR）受到了更多的关注。虽然蛋白酶依赖于肝素结合生长因子的脱落，但在这种情况下，肝素结合表皮生长因子（HB-EGF）似乎对EGFR的凝血酶反式激活负有负责，但我们对这种EGFR的反应影响促进症诱导的VSMC Miteason和Motogensogens and Ampactactivativativation GAP似乎存在差距。主要问题是，EGFR的反式激活是否足以刺激受体下游的有丝分裂和/或MOTOGONIC信号流动？为了阐明这些信号流，我们发现凝血酶激活了与GRB2相关的结合蛋白1（GAB1）和SRC同源性2-含有蛋白质酪氨酸磷酸酶（SHP2），其刺激是另有预期的刺激，其响应于EGFR激活其真实的配体EGF，EGF的刺激，EGF刺激了该功能性的EGGFR。更令人兴奋的是，凝血酶诱导的Rho GTPase刺激和F-肌动蛋白应力纤维形成需要GAB1-SHP2激活。基于这些新颖的观察，我们建议测试以下特定目标，其目标是阐明G蛋白耦合受体（GPCR）信号流，这些信号流是在人主动脉平滑肌细胞（HASMC）中向上和下游到EGFR反式激活的EGFR反式激活（HASMC）（HASMC）中的EGFR反式激活，并测试其在f-act纤维形成中的强度，并测试f-Actifer contratife and Felro的介导，并在迁移型纤维中的介导，并在迁移依次的介导，并将其迁移效果供应供应，并在迁移依次的介导，从而在迁移式纤维中，从而介导了源自源的介导，并将其与临床的介导相结合，从体内血管成形术后的重塑。在本研究建议中将解决的具体目的如下：1。凝血酶诱导的HASMC F-肌动蛋白应力纤维形成，迁移，增殖和新内膜形成需要GAB1激活。 2。凝血酶通过GAB1和Rhogef依赖性RhoA，Rac1和Cdc42激活介导Hasmc F-肌动蛋白应力纤维的形成，迁移，增殖和新主体形成来激活Rho GTPases。 3。GAB1靶向PAK1介导凝血酶诱导的HASMC F-肌动蛋白应力纤维形成，迁移，增殖和新内膜形成。简而言之，提出的实验的结果将填补我们对GPCR信号通过RTK信号传导如何通过rtk信号传导和靶向脚手架适配器分子GAB1的差距的空白，从而导致Rhogef介导的Rhoa-Rac1/cdc42-Pak1激活在血管壁重塑后在血管壁重塑后起作用。关于血管壁疾病病理生物学的这种全面知识可能成为控制这些血管病变药物的有价值的工具。 公共卫生相关性：血管平滑肌细胞（VSMC）迁移和增殖在血管成形术或静脉移植后的动脉粥样硬化和再狭窄等外周血管疾病中起重要作用。阐明VSMC迁移和增殖的分子机制对于控制这些血管病变的疾病过程的发展至关重要。在这方面，目前的赠款提案旨在研究凝血酶，G蛋白偶联受体激动剂和血管损伤部位产生的凝血因子的机制，可以反式激活受体酪氨酸激酶信号在刺激VSMC迁移和增殖和点燃疾病过程中刺激。