Mechanism for feedback regulation of G protein-coupled receptor signaling in platelets

血小板G蛋白偶联受体信号反馈调节机制

基本信息

批准号：
10181028
负责人：
Peisong Ma
金额：
$ 39万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10181028
关键词：
ADRBK1 gene Address Adrenergic Agents African American Agonist Binding Binding Proteins Biochemistry Blood Platelets CRISPR/Cas technology Cardiovascular Diseases Cardiovascular Pathology Cardiovascular system Cells Cerebrovascular Disorders Clustered Regularly Interspaced Short Palindromic Repeats Data Ensure Environment Event Family Feedback G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors GRK5 gene GRK6 gene GTP-Binding Protein Regulators GTP-Binding Proteins Goals Health Heart Heart Hypertrophy Hematology Hemostatic function Human Hypertension Inflammation Injury Integrins Knock-out Knowledge Lead Left Link Modeling Morbidity - disease rate Mus Mutant Strains Mice Mutation Myocardial Infarction Phospholipase C Phosphotransferases Platelet Activation Play Protein Kinase RGS Proteins Regulation Resistance Rest Role Signal Transduction Signaling Protein Single Nucleotide Polymorphism Site Stroke Surface Testing Thrombosis Thrombus Time Tissues Transcript United States Variant Work base cardiovascular health cell type cerebrovascular genetic variant genome wide association study in vivo induced pluripotent stem cell innovation mutant novel novel therapeutics platelet function prevent public health relevance receptor response response to injury therapeutic target thrombotic vascular injury

项目摘要

Achieving hemostasis following vascular injury while avoiding excessive platelet accumulation requires that signaling is closely regulated in resting and activated platelets. Most platelet agonists work through G protein coupled receptors (GPCRs). Our goals are to dissect how GPCRs and G proteins can be regulated by GPCR kinases (GRKs) during platelet activation and thrombus formation, and to understand how dysfunctional regulation of GRKs may lead to thrombotic events and cardiovascular disease. In the past two decades, GRKs have been shown to play an important role in the heart by regulating GPCR signaling. Changes in GRK expression have been linked to many cardiovascular pathologies. However, the contribution of GRKs to platelet activation and the role of GRKs in hemostasis and thrombosis are unknown. Our hypothesis is that GRKs are critical negative regulators of platelet activation and thrombus formation. We base our hypothesis on preliminary studies from our lab showing that 1) The RGS-resistant G188S mutation in Gq prevents RGS protein binding but surprisingly increases GRK2 binding; 2) GRK2 binding is specific to Gq, but not to Gi2; 3) In contrast to enhanced Gi2 signaling in RGS-resistant Gi2(G184S) mutant platelets, there is decreased platelet activation in Gq(G188S) platelets; 4) Deletion of GRK2, GRK5 or GRK6 in platelets causes an increase in platelet activation. The hypothesis will be tested in three specific aims. In Aim 1, we will examine the role of G protein and GRKs interactions in regulating platelet function. In Aim 2, we will determine the role of GRK2 in hemostasis and thrombosis. we will be the first to characterize the functions of GRK2 in hemostasis and thrombosis and identify the non-canonical roles of GRK2 in platelets. In Aim 3, we will determine the role of GRK5/6 in hemostasis and thrombosis and explore the effect of two human GRK5 genetic variants on platelet function using CRISPR-Cas9 edited iPSCs (induced pluripotent stem cells). These proposed studies are innovative because we combine recent genome-wide association studies (GWAS) identified human GRK5 genetic variants, 4 newly generated mouse mutant lines and CRISPR-cas9 edited iPSC cells to study the uncharacterized role of GRKs in platelets. This study is significant because a critical gap in knowledge exists between unexplored functions of GRK family in platelets and their well-studied roles in the cardiovascular system. Through these studies, we will advance our understanding of the role of GRKs in cardiovascular health and disease, and the gained information may lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.

血管损伤后实现止血，同时避免血小板过度积聚需要在静息和活化血小板中密切调节信号传导。大多数血小板激动剂通过 G 蛋白偶联受体 (GPCR) 发挥作用。我们的目标是剖析如何 GPCR 和 G 蛋白在血小板激活过程中可以受到 GPCR 激酶 (GRK) 的调节和血栓形成，并了解 GRK 的失调调节如何可能导致血栓事件和心血管疾病。在过去的二十年里，GRK 通过调节 GPCR 信号传导在心脏中发挥重要作用。 GRK 的变化表达与许多心血管病理有关。然而，贡献 GRKs 对血小板的激活以及 GRKs 在止血和血栓形成中的作用尚不清楚。我们的假设是 GRK 是血小板活化和血栓的关键负调节因子形成。我们的假设基于我们实验室的初步研究表明：1) Gq 中的 RGS 抗性 G188S 突变可阻止 RGS 蛋白结合，但令人惊讶地增加 GRK2 结合； 2) GRK2 结合对 Gq 具有特异性，但对 Gi2 没有特异性； 3）与增强型Gi2相比 RGS 抗性 Gi2(G184S) 突变血小板中的信号传导，在 Gq(G188S)血小板； 4) 血小板中GRK2、GRK5或GRK6的缺失会导致血小板增多血小板活化。该假设将在三个具体目标上进行检验。在目标 1 中，我们将检查 G 蛋白和 GRK 相互作用在调节血小板功能中的作用。在目标 2 中，我们将确定GRK2在止血和血栓形成中的作用。我们将首先描述 GRK2 在止血和血栓形成中的功能，并确定其非典型作用血小板中的GRK2。在目标 3 中，我们将确定 GRK5/6 在止血和血栓形成中的作用并利用两种人类 GRK5 基因变异探索血小板功能的影响 CRISPR-Cas9 编辑 iPSC（诱导多能干细胞）。这些拟议的研究是创新是因为我们结合了最近确定的全基因组关联研究 (GWAS) 人类 GRK5 遗传变异、4 个新生成的小鼠突变系和 CRISPR-cas9 编辑 iPSC 细胞研究 GRK 在血小板中的未表征的作用。这项研究意义重大因为 GRK 家族的未探索功能之间存在着重大的知识差距血小板及其在心血管系统中的作用得到充分研究。通过这些研究，我们将增进我们对 GRK 在心血管健康和疾病中作用的理解，并且所获得的信息可能会导致治疗血栓和血栓性疾病的新治疗选择心血管疾病。