The structural basis for PAR1 biased signaling

项目摘要/摘要 G蛋白偶联受体通过激活GαQ、Gα12/13、 GαI，或arrestin。人们越来越认识到，有偏见的激动剂可以决定哪些信号通路是 在受体下游被激活。蛋白水解酶激活受体(PARs)是 蛋白水解酶启动细胞内信号传递。PARs被N-末端的裂解激活，以产生系链 莱兰德。PAR1对多个蛋白水解酶具有独特的切割位点，可导致一组独特的系留配体。 这些配体是内源性偏向激动剂，可以触发特定的信号通路。基因的分子基础 这还不为人所知。该研究计划长期目标是定义解析器如何调解特定的上下文 内皮细胞、血小板和其他细胞中的信号转导。该项目旨在为 了解控制正常生理反应的PAR激活机制的分子基础。 这项提案的总体目标是1.)确定PAR1的拴系配体结合位点(S) 三种内源性激活剂凝血酶、APC和MMP12。)揭示PAR1偏向的结构基础 信令3。)确定这些位置如何协作来调节特定的生理信号事件。我们的整体 假设PAR1采用特定的构象，因为每个拴系的配体结合位置不同 决定哪些信号通路被激活的配体。科学前提是建立在最近的成功基础上的 我们的实验设计结合了酰胺氢/氢(H/D)交换和纯化的部分 确定连接配体如何影响整体构象。分子模拟将独立进行 确定配体结合部位(S)。最后，确定的区域将在细胞信号分析中使用一个小组进行测试 以验证PAR1突变体在细胞信号转导中的重要性。我们的创新方法将确定以前的 凝血酶、APC和APC产生的每个拴系配体的内源性配体结合位点未知 MMP1。这些研究完成后，将确定PAR1与内源的潜在构象 激活剂。这些实验将提供关于单个受体如何能够 在正常生理条件下，相反的信号结果。

PROJECT SUMMARY/ABSTRACT G-protein coupled receptors (GPCRs) elicit complex downstream signaling cascades by activating Gαq, Gα12/13, Gαi, or arrestin. There is a growing appreciation that biased agonists can dictate which signaling pathways are activated downstream of the receptor. Protease activated receptors (PARs) are the primary means by which proteases initiate intracellular signaling. PARs are activated by cleavage of the N-terminus to generate a tethered ligand. PAR1 has unique cleavage sites for multiple proteases that can lead to a panel of unique tethered ligands. These ligands are endogenous biased agonists that trigger specific signaling pathways. The molecular basis for this is not known. The long-term goals of this research program are to define how PARs mediate context specific signaling in endothelial cells, platelets and other cells. This project seeks to develop a foundation for understanding the molecular basis for PAR activation mechanisms that govern normal physiological responses. The overall objective of this proposal is to 1.) define the tethered ligand binding site(s) for PAR1 activated by three endogenous activators thrombin, APC, and MMP1 2.) uncover the structural basis for PAR1 biased signaling 3.) determine how these sites cooperate to mediate specific physiological signaling events. Our overall hypothesis is that PAR1 adopts specific conformations due to distinct ligand binding sites for each of the tethered ligands dictating which signaling pathways are activated. The scientific premise is based on the recent success of our experimental design that incorporates amide hydrogen/deuterium (H/D) exchange with purified PARs to determine how the tethered ligand influences the overall conformation. Molecular modeling will independently determine the ligand binding site(s). Finally, identified regions will be tested in cell signaling assays using a panel of PAR1 mutants to verify the importance on cell signaling. Our innovative approach will identify the previously unknown endogenous ligand binding sites for each of the tethered ligands generated by thrombin, APC, and MMP1. At the completion of these studies will define the potential conformations of PAR1 with endogenous activators. These experiments will provide the first structural insights as to how a single receptor can have opposite signaling outcomes under normal physiological conditions.