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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：
10241452
负责人：
Marvin Thomas Nieman
金额：
$ 20.13万
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 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241452
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.

项目总结/摘要 G蛋白偶联受体（GPCR）通过激活Gαq，Gα12/13， Gαi或抑制蛋白。越来越多的人认识到，偏向性激动剂可以决定哪些信号通路是在受体下游被激活。蛋白酶激活受体（PAR）是主要的手段，蛋白酶启动细胞内信号传导。PAR通过N-末端的切割被激活以产生栓系的配体。PAR 1具有多种蛋白酶的独特切割位点，其可导致一组独特的拴系配体。这些配体是触发特定信号传导途径的内源性偏向性激动剂。的分子基础这是未知的。本研究计划的长期目标是确定PAR如何介导特定环境内皮细胞、血小板和其他细胞中的信号传导。该项目旨在建立一个基础，了解PAR激活机制的分子基础，控制正常的生理反应。本提案的总体目标是：（1）定义PAR 1的栓系配体结合位点，三种内源性激活剂凝血酶、APC和MMP 1 2）。揭示PAR 1偏向的结构基础信令3.）确定这些位点如何合作来介导特定的生理信号事件。我们的整体假设是PAR 1由于每个拴系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在我们的实验设计中，将酰胺氢/氘（H/D）交换与纯化的PAR结合，确定束缚的配体如何影响整体构象。分子建模将独立地确定配体结合位点。最后，将在细胞信号传导测定中使用一组 PAR 1突变体，以验证对细胞信号传导的重要性。我们的创新方法将确定以前的由凝血酶、APC和THP产生的每个拴系配体的未知内源性配体结合位点， MMP1。在这些研究完成时，将确定PAR 1与内源性活化剂。这些实验将提供关于单个受体如何具有在正常生理条件下的相反信号结果。