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，除了在血液凝固中起作用外，还作为许多细胞类型的有丝分裂原和运动原发挥重要作用，特别是对血管平滑肌细胞（VSMCs）。凝血酶通过蛋白酶激活受体（PARs），即PAR-1、PAR-2、PAR-3和PAR-4介导其作用。在PARs下游，它需要G蛋白的参与，特别是Gq/11或G12/13参与细胞迁移和增殖的调节。此外，凝血酶具有反激活酪氨酸受体激酶（RTKs）的能力，其中表皮生长因子受体（EGFR）受到较多关注。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反激活，但我们对这种EGFR反激活如何影响凝血素诱导的VSMC有丝分裂和运动发生的理解似乎存在差距。主要的问题是，EGFR的反激活是否足以刺激有丝分裂和/或运动信号流向下游的受体？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2的蛋白酪氨酸磷酸酶（Shp2），其刺激是在其真正的配体EGF激活EGFR时预期的，并且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，Gab1-Shp2激活是凝血素诱导的Rho GTPase刺激和f -肌动蛋白应激纤维形成所必需的。基于这些新的观察结果，我们提出测试以下特定目标，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 -肌动蛋白应激纤维形成、迁移和增殖的介导强度，以响应凝血酶和血管成形术后血管壁重塑。在这个研究计划中，具体的目标是：1。凝血酶诱导的HASMC f -肌动蛋白应激纤维的形成、迁移、增殖和新生内膜的形成都需要Gab1的激活。2. 凝血酶通过Gab1和rhogef依赖性RhoA募集rhogef激活Rho gtpase， Rac1和Cdc42激活介导HASMC F-actin应激纤维的形成、迁移、增殖和新生内膜的形成。3. Gab1靶向PAK1介导凝血酶诱导的HASMC F-actin应激纤维的形成、迁移、增殖和新生内膜的形成。简而言之，这些实验的结果将填补我们对GPCR信号如何通过与RTK信号的串扰并靶向支架接头分子Gab1，导致rhogef介导的RhoA-Rac1/Cdc42-PAK1激活在损伤后血管壁重塑中发挥作用的理解空白。这些关于血管壁疾病病理生物学的全面知识可以成为开发控制这些血管病变的药物的宝贵工具。

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.