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.

项目概要/摘要 G 蛋白偶联受体 (GPCR) 通过激活 Gαq、Gα12/13、人们越来越认识到偏向激动剂可以决定哪些信号通路。受体下游的蛋白酶激活受体（PAR）是激活的主要手段。蛋白酶启动细胞内信号传导，通过 N 末端的切割来激活，产生束缚。 PAR1 具有针对多种蛋白酶的独特切割位点，可产生一组独特的束缚配体。这些配体是触发特定信号通路的内源性偏向激动剂。这个研究项目的长期目标是定义 PAR 如何调节特定环境。该项目旨在为内皮细胞、血小板和其他细胞中的信号传导奠定基础。了解控制正常生理反应的 PAR 激活机制的分子基础。该提案的总体目标是 1.) 定义由以下物质激活的 PAR1 的系留配体结合位点：三种内源性激活剂凝血酶、APC 和 MMP1 2.) 揭示 PAR1 偏向性的结构基础信号传导 3.) 确定这些位点如何合作来介导我们整体的特定生理信号传导事件。假设 PAR1 由于每个束缚的配体结合位点不同而采用特定的构象配体决定哪些信号通路被激活，科学前提是基于最近的成功。我们的实验设计将酰胺氢/氘 (H/D) 与纯化的 PAR 交换结合起来确定束缚配体如何影响整体构象将独立进行。确定配体结合位点最后，将使用面板在细胞信号转导测定中测试已识别的区域。 PAR1 突变体以验证对细胞信号传导的重要性。凝血酶、APC 和生成的每个束缚配体的未知内源配体结合位点这些研究完成后将确定 PAR1 与内源性的潜在构象。这些实验将提供关于单个受体如何具有的第一个结构见解。正常生理条件下相反的信号结果。