GPCR Signaling & Vascular Wall Remodeling

说明(申请人提供)：凝血酶是一种丝氨酸/苏氨酸蛋白酶，除了在凝血中起作用外，对许多细胞，特别是血管平滑肌细胞(VSMCs)，它也是一种有丝分裂原和运动原。凝血酶通过蛋白水解酶激活受体(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，凝血酶还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。在这种情况下，肝素结合生长因子(HB-EGF)的蛋白酶依赖的脱落似乎是导致EGFR凝血酶反式激活的原因，但我们对这种EGFR反式激活如何影响凝血酶诱导的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的反式激活是否足以刺激下游至受体的有丝分裂和/或动生信号流？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2的蛋白酪氨酸磷酸酶(Shp2)，而Shp2的刺激是通过其真正的配体EGF来响应EGFR的激活，而这种多功能信号复合体的刺激需要EGFR酪氨酸激酶的活性。更令人兴奋的是，凝血酶诱导的Rho GTP酶刺激和F-肌动蛋白应激纤维的形成需要GAB1-Shp2的激活。基于这些新的观察结果，我们建议测试以下特定的目标，目的是阐明人主动脉平滑肌细胞(HASMCs)中EGFR反式激活的G蛋白偶联受体(GPCR)信号流，并测试它们在介导这些细胞在体外对凝血酶反应的F-肌动蛋白应激纤维形成、迁移和增殖以及体内血管成形术后血管壁重建中的强度。本研究的具体目标如下：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需要GAB1的激活。2.凝血酶通过GAB1募集RhoGEF激活Rho GTP酶，RhoGEF依赖的RhoA、rac1和CDC42激活介导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。3.GAB1靶向PAK1介导凝血酶诱导的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。简而言之，拟议的实验结果将填补我们对GPCR信号如何通过串扰与RTK信号并靶向支架适配器分子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上的空白。这种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有价值的工具。

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.