GPCR Signaling & Vascular Wall Remodeling

描述（由申请人提供）：凝血酶是一种丝氨酸/苏氨酸蛋白酶，除了其在血液凝固中的作用外，还作为许多细胞类型，特别是血管平滑肌细胞（VSMC）的有丝分裂原和运动原发挥重要作用。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。在PAR下游，它需要G蛋白，特别是Gq/11或G12/13参与细胞迁移和增殖的调节。此外，凝血酶还具有反式激活受体酪氨酸激酶（receptor tyrosine kinases，RTKs）的能力，其中表皮生长因子受体（epidermal growth factor receptor，EGFR）受到了广泛的关注。虽然肝素结合生长因子的蛋白酶依赖性脱落，在这种情况下，肝素结合表皮生长因子（HB-EGF），似乎是负责凝血酶的EGFR反式激活，似乎有一个差距，在我们的理解，这种EGFR反式激活如何影响凝血酶诱导的血管平滑肌细胞有丝分裂和运动。主要的问题是，EGFR的反式激活是否足以刺激有丝分裂和/或运动信号流向受体下游？为了阐明这些信号流，我们发现，凝血酶激活GRB 2相关的结合蛋白1（Gab 1）和Src同源性2-含有蛋白酪氨酸磷酸酶（Shp 2），其刺激，否则预期在EGFR激活其真正的配体，EGF，和刺激这种多功能信号复合物需要EGFR酪氨酸激酶活性。更令人兴奋的是，Gab 1-Shp 2激活是凝血酶诱导的Rho GT3刺激和F-actin应力纤维形成所必需的。基于这些新的观察结果，我们建议测试以下特定目标，目的是阐明人主动脉平滑肌细胞（HASMCs）中EGFR反式激活的上游和下游G蛋白偶联受体（GPCR）信号流，并测试其在F-肌动蛋白应力纤维形成中的介导强度，这些细胞在体外响应凝血酶的迁移和增殖以及在体内血管成形术后的血管壁重塑。本研究的具体目标如下：1.凝血酶诱导的HASMC F-actin应力纤维形成、迁移、增殖和新生内膜形成需要Gab 1激活。2.凝血酶通过由Gab 1和RhoGEF依赖性RhoA、Rac 1和Cdc 42激活的Rho GTPases募集RhoGEF来激活Rho GTPases，从而介导HASMC F-actin应力纤维形成、迁移、增殖和新生内膜形成。3. Gab 1靶向PAK 1介导凝血酶诱导的HASMC F-actin应力纤维形成、迁移、增殖和新生内膜形成。简而言之，所提出的实验的结果将填补我们理解GPCR信号传导如何通过与RTK信号传导的串扰并靶向支架衔接分子Gab 1，导致RhoGEF介导的RhoA-Rac 1/Cdc 42-PAK 1活化在损伤后的血管壁重塑中发挥作用的差距。血管壁疾病的病理生物学的这种全面的知识可能成为一个有价值的工具，在开发药物控制这些血管病变。

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.