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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：
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.

项目总结/文摘