Regulation of vascular permeability by thrombin mediated signaling pathways

描述(由申请人提供)：本方案的目标是了解单个受体下游多个G蛋白通路的信号整合。我们建议建立多个可计算的“信号模块”，代表凝血酶受体PAR1下游的G蛋白信号通路。已知PAR1通过与包括Gi、Gq和G12/13家族成员在内的多个G蛋白偶联，从而激活这些信号通路来调节内皮屏障功能，从而介导其在内皮细胞中的复杂作用。为G蛋白信号通路建模奠定的数学基础还没有被应用于这样的多通路整合问题。血管内皮细胞(EC)在血液和间质组织之间形成一道动态调节的屏障，凝血酶是屏障通透性最强的调节因子。屏障功能障碍会导致水肿，这是创伤和炎症的正常后果，但如果不加以控制，则会导致许多疾病，如急性肺损伤和脓毒症。我们的目标是1)构建一个数学和计算模型，描述PARS下游的多个G蛋白信号通路，作为系统研究调节内皮通透性的详细分子机制的工具。2)确定单个G蛋白参与生理反应的机制。该实验室已经开发出创新的工具，一次解开一条G蛋白途径，并确定对细胞反应的影响。3)使用该模型帮助确定将成为这些疾病治疗干预的新靶点的关键部位。利用模型的早期迭代，我们发现凝血酶和凝血酶受体激活肽对不同类别的G蛋白信号通路有不同的调节作用。我们将研究不同构象的PAR1向不同的G蛋白传递的假说，并对屏障功能进行差异调节。这将提供一个治疗窗口，以寻找作用于不同于系留配体的PARs的变构调节剂。在整个赠款过程中，建模、仿真和实验将反复进行。长期目标是建立一个用于凝血酶调节内皮屏障的复杂信号过程的有效计算模型。该模型将有助于确定关键部位，这些部位将成为这些疾病治疗干预的新目标。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand signal integration of multiple G protein pathways downstream of a single receptor. We propose to build up multiple computable "signaling modules", which represent G protein signaling pathways downstream of a multiply coupled receptor, PAR1, the thrombin receptor. PAR1 is known to mediate its complex actions in endothelium via its ability to couple to multiple G proteins including members of Gi, Gq and G12/13 families thus leading to activation of these signaling pathways to modulate endothelial barrier function. The mathematical ground work laid in modeling of G protein signaling pathways has not yet been applied to such a multiple pathway integration problem. Endothelial cells (EC) form a dynamically regulated barrier between blood and interstitial tissues; thrombin is the most potent regulator of barrier permeability. Barrier dysfunction leads to edema, a normal consequence of wounding and inflammation, but when uncontrolled is a devastating component of many diseases such as acute lung injury and sepsis. Our aims are to 1) Construct a mathematical and computational model that describes the multiple G protein signaling pathways downstream of PARs that will serve as a tool to systematically investigate the detailed molecular mechanisms that regulate endothelial permeability. 2) Determine the mechanisms by which individual G proteins contribute to the physiological response. The laboratory has developed innovative tools to uncouple one G protein pathway at a time and determine the effects on cellular responses. 3) Use the model to help to identify key sites that would be novel targets for therapeutic intervention in these disorders. Using an early iteration of the model, we showed that thrombin and the thrombin receptor activating peptide differentially regulate different classes of G protein signaling pathways. We will investigate the hypothesis that different conformations of PAR1 traffic to different G proteins, and differentially regulate barrier function. This would provide a therapeutic window to search for allosteric modulators of PARs that work at different sites than the tethered ligand. Throughout the grant, the modeling, simulations and experimentation will be conducted iteratively. The long term goals are to build toward production of a validated computational model of the complex signaling processes used in thrombin regulation of the endothelial barrier. The model will help to identify key sites that would be novel targets for therapeutic intervention in these disorders.