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

在血管损伤后实现止血，同时避免血小板过度聚集 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）起作用。我们的目标是剖析 血小板活化过程中GPCR和G蛋白可受GPCR激酶（GRKs）的调节 和血栓形成，并了解GRKs的功能失调如何导致 血栓形成事件和心血管疾病。在过去的二十年里，GRK一直是 通过调节GPCR信号在心脏中发挥重要作用。GRK的变化 表达与许多心血管疾病有关。然而， GRK对血小板活化的作用以及GRK在止血和血栓形成中的作用尚不清楚。 我们的假设是GRKs是血小板活化和血栓形成的关键负调节因子 阵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G188 S突变阻止RGS蛋白结合，但令人惊讶地增加了RGS蛋白结合。 GRK 2结合; 2）GRK 2结合特异于Gq，但不特异于Gi 2; 3）与增强的Gi 2相反， 在RGS抗性Gi 2（G184 S）突变体血小板中， Gq（G188 S）血小板; 4）血小板中GRK 2、GRK 5或GRK 6的缺失导致血小板中GRK 2、GRK 5或GRK 6的增加。 血小板活化该假设将在三个具体目标中进行检验。在目标1中，我们将研究 G蛋白和GRKs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GRK 2在止血和血栓形成中的作用。我们将是第一个描述 GRK 2在止血和血栓形成中的功能，并确定GRK 2的非经典作用。 血小板中的GRK 2。在目标3中，我们将确定GRK 5/6在止血和血栓形成中的作用。 并使用免疫组织化学方法研究两种人类GRK 5基因变异体对血小板功能的影响。 CRISPR-Cas9编辑的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联合收割机最近全基因组关联研究（GWAS）确定 人类GRK 5遗传变体，4种新产生的小鼠突变系和CRISPR-cas9编辑 iPSC细胞来研究GRKs在血小板中的未表征作用。这项研究意义重大 因为在GRK家族的未开发功能之间存在着关键的知识差距， 血小板及其在心血管系统中充分研究的作用。通过这些研究，我们 将促进我们对GRKs在心血管健康和疾病中作用的理解， 所获得的信息可能导致治疗血栓形成的新的治疗选择， 心血管疾病

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.