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

• 批准号: 10415099
• 负责人: Peisong Ma
• 金额: $ 39万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415099
• 关键词: ADRBK1 gene; Address; Adrenergic Agents; African American population; 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

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.

血管损伤后达到止血，同时避免过度血小板积累 要求信号在静止和激活的血小板中受到密切调节。最血小板 激动剂通过G蛋白偶联受体（GPCR）进行工作。我们的目标是剖析 在血小板激活过程中，GPCR和G蛋白可以由GPCR激酶（GRKS）调节 和血栓形成，并了解GRK功能失调的调节可能导致 血小板事件和心血管疾病。在过去的二十年中，格克斯一直 通过调节GPCR信号传导，显示出在心脏中起重要作用。 GRK的变化 表达与许多心血管病理有关。但是， GRK到血小板激活以及GRK在止血和血栓形成中的作用尚不清楚。 我们的假设是GRK是血小板激活和血栓的关键负调节剂 形成。我们将假设以实验室的初步研究为基础，表明1） GQ中耐RGS的G188S突变可防止RGS蛋白结合，但令人惊讶的是增加 grk2结合； 2）grk2结合特定于GQ，但不针对GI2； 3）与增强的GI2相反 耐RGS的GI2（G184S）突变体血小板中的信号传导，血小板激活降低 GQ（G188S）血小板； 4）血小板中删除GRK2，GRK5或GRK6会导致增加 血小板激活。该假设将以三个特定目的进行检验。在AIM 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AIM 2中，我们将 确定GRK2在止血和血栓形成中的作用。我们将是第一个描述的人 GRK2在止血和血栓形成中的功能，并确定 血小板中的GRK2。在AIM 3中，我们将确定GRK5/6在止血和血栓形成中的作用 并探索使用两个人Grk5遗传变异对血小板功能的影响 CRISPR-CAS9编辑了IPSC（诱导多能干细胞）。这些提出的研究是 创新性是因为我们结合了鉴定出的最新基因组关联研究（GWAS） 人类GRK5遗传变异，4种新生成的小鼠突变线和CRISPR-CAS9编辑 IPSC细胞研究GRK在血小板中的未表征作用。这项研究很重要 因为在GRK家族的未探索功能之间存在关键的知识差距 血小板及其在心血管系统中的良好作用。通过这些研究，我们 将促进我们对GRK在心血管健康和疾病中的作用的理解，以及 获得的信息可能会导致新的治疗选择，以治疗血栓形成和 心血管疾病。

项目成果

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12.

• DOI: 10.1182/bloodadvances.2022007007
• 发表时间: 2022-08-09
• 期刊: BLOOD ADVANCES
• 影响因子: 7.5
• 作者: Zhao, Xuefei;Cooper, Matthew;Michael, James V.;Yarman, Yanki;Baltz, Aiden;Chuprun, J. Kurt;Koch, Walter J.;McKenzie, Steven E.;Tomaiuolo, Maurizio;Stalker, Timothy J.;Zhu, Li;Ma, Peisong
• 通讯作者: Ma, Peisong

G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1.

• DOI: 10.1182/bloodadvances.2021005453
• 发表时间: 2022-04-12
• 期刊: BLOOD ADVANCES
• 影响因子: 7.5
• 作者: Downes, Kate;Zhao, Xuefei;Gleadall, Nicholas S.;McKinney, Harriet;Kempster, Carly;Batista, Joana;Thomas, Patrick L.;Cooper, Matthew;Michael, James, V;Kreuzhuber, Roman;Wedderburn, Katherine;Waller, Kathryn;Varney, Bianca;Verdier, Hippolyte;Kriek, Neline;Ashford, Sofie E.;Stirrups, Kathleen E.;Dunster, Joanne L.;McKenzie, Steven E.;Ouwehand, Willem H.;Gibbins, Jonathan M.;Yang, Jing;Astle, William J.;Ma, Peisong
• 通讯作者: Ma, Peisong