GPCR Signaling & Vascular Wall Remodeling

GPCR 信号转导

基本信息

批准号：
8444697
负责人：
GADIPARTHI N RAO
金额：
$ 44.1万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
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/8444697
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 public health relevance receptor research study response restenosis rho GTP-Binding Proteins scaffold tool vascular smooth muscle cell migration

项目摘要

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.

描述（由申请人提供）：凝血酶是一种丝氨酸/苏氨酸蛋白酶，除了在血液凝固中的作用之外，对于许多细胞类型，特别是血管平滑肌细胞（VSMC），作为有丝分裂原和促生长素也发挥着重要作用。凝血酶通过蛋白酶激活受体 (PAR)（即 PAR-1、PAR-2、PAR-3 和 PAR-4）介导其作用。 PAR 的下游需要 G 蛋白的参与，特别是 Gq/11 或 G12/13 来调节细胞迁移和增殖。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）受到更多关注。虽然肝素结合生长因子（在本例中为肝素结合表皮生长因子 (HB-EGF)）的蛋白酶依赖性脱落似乎与 EGFR 的凝血酶反式激活有关，但我们对这种现象如何发生的理解似乎存在差距。 EGFR 反式激活影响凝血酶诱导的 VSMC 有丝分裂和运动发生。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/或运动信号流？为了阐明这些信号流，我们发现凝血酶激活 GRB2 相关结合蛋白 1 (Gab1) 和含有 Src 同源性 2 的蛋白酪氨酸磷酸酶 (Shp2)，其刺激预计是对其真正配体 EGF 激活 EGFR 的反应，而这种多功能信号复合物的刺激需要 EGFR 酪氨酸激酶活性。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。基于这些新的观察结果，我们建议测试以下具体目标，旨在阐明人主动脉平滑肌细胞（HASMC）中 EGFR 反式激活的上游和下游的 G 蛋白偶联受体（GPCR）信号流，并测试其体外对凝血酶的响应以及体内血管成形术后血管壁重塑中这些细胞的 F-肌动蛋白应力纤维形成、迁移和增殖的介导强度。本研究计划要解决的具体目标如下： 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 招募 RhoGEF 来激活 Rho GTPases，RhoGEF 依赖性 RhoA、Rac1 和 Cdc42 激活介导 HASMC F-肌动蛋白应力纤维形成、迁移、增殖和新内膜形成。 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，所提出的实验结果将填补我们对 GPCR 信号如何通过与 RTK 信号串扰并靶向支架适配器分子 Gab1 的理解空白，从而导致 RhoGEF 介导的 RhoA-Rac1/Cdc42-PAK1 激活在损伤后血管壁重塑。这种关于血管壁疾病病理学的全面知识可能成为开发控制这些血管病变的药物的宝贵工具。