The structural basis for PAR1 biased signaling

PAR1 偏向信号传导的结构基础

基本信息

批准号：
10042725
负责人：
Marvin Thomas Nieman
金额：
$ 24.15万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10042725
关键词：
Address Adopted Agonist Amides Arrestins Binding Sites Biological Biological Assay Blood Platelets Cells Charge Cleaved cell Complex Data Data Analyses Deuterium Endothelial Cells Event Experimental Designs Foundations G-Protein-Coupled Receptors Goals Hydrogen Interstitial Collagenase Lead Ligand Binding Ligands Mass Spectrum Analysis Mediating Mission Molecular Molecular Conformation Molecular Target Mutation Analysis Outcome PAR-1 Receptor PAWR gene Pathologic Pathway interactions Peptide Hydrolases Physiological Positioning Attribute Proteinase-Activated Receptors Public Health Receptor Activation Research Resolution Signal Pathway Signal Transduction Site Structure Techniques Technology Testing Therapeutic Thrombin United States National Institutes of Health base experience experimental study extracellular innovation insight interest molecular modeling mutant novel novel strategies programs receptor receptor function response structural biology success

项目摘要

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.

项目摘要/摘要 G蛋白偶联受体（GPCR）通过激活GαQ，Gα12/13，引起下游信号级联 GαI或逮捕。越来越多的欣赏是偏见的激动剂可以决定哪些信号通路是受体的下游激活。蛋白酶活化受体（PAR）是主要手段蛋白酶启动细胞内信号传导。 N-末端的裂解激活PAR，以产生束缚配体。 PAR1具有用于多种蛋白酶的独特切割位点，可以导致一系列独特的系带配体。这些配体是触发特定信号通路的内源性偏见激动剂。分子基础这是不知道的。该研究计划的长期目标是定义PAR如何介导环境特定环境内皮细胞，血小板和其他细胞中的信号传导。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。该提案的总体目的是1.）定义由PAR1激活的束缚配体结合位点三个内源激活剂凝血酶，APC和MMP1 2.）发现PAR1的结构基础有偏见信号3.）确定这些位点如何坐标以介导特定的物理信号事件。我们的整体假设是，PAR1由于每个束缚的配体结合位点而采用特定的考虑因素指示哪种信号通路激活的配体。科学前提是基于最近的成功我们的实验设计，该设计结合了酰胺氢/氘（H/D）交换，并纯化确定束缚配体如何影响整体考虑。分子建模将独立确定配体结合位点。最后，将使用面板在细胞信号测定中测试确定的区域 PAR1突变体以验证细胞信号的重要性。我们的创新方法将确定以前的凝血酶，APC和 MMP1。这些研究完成时，将定义PAR1的潜在构象与内生的构象激活剂。这些实验将提供有关单个接收器如何具有的第一个结构见解在正常生理条件下的相反信号传导结果。