Defining the Role of of Noncanonical GPCR Signalling in Pulmonary Hypertension

定义非典型 GPCR 信号在肺动脉高压中的作用

基本信息

批准号：
10669247
负责人：
Claudia Y Lee
金额：
$ 3.86万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10669247
关键词：
Angiotensin II Receptor Biochemical Biological Assay Biotin CXCR Cardiovascular system Cell Line Cell physiology Complex Coupling Development Disease Drug Targeting Drug usage Endosomes Endothelial Cells Endothelium Engineering Event Extracellular Signal Regulated Kinases FDA approved G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Genome Goals Harvest Health Heterotrimeric GTP-Binding Proteins Impairment Knowledge Label Ligands Medical Mission Mutate Pathway interactions Patients Peroxidases Pharmaceutical Preparations Physiological Physiology Play Proliferating Proteins Pulmonary Hypertension Receptor, Angiotensin, Type 1 Research Role Scientist Signal Pathway Signal Transduction System Testing Therapeutic Training Tube United States National Institutes of Health Variant Vascular remodeling Work ascorbate beta-arrestin chemokine receptor desensitization endothelial dysfunction insight migration nanoluciferase novel novel therapeutics pharmacologic preference public health relevance pulmonary artery endothelial cell pulmonary vascular disorder receptor receptor function receptor internalization right ventricular failure tool trafficking vasoconstriction

项目摘要

ABSTRACT Pulmonary hypertension (PH) is characterized by endothelial dysfunction, irregular vascular remodeling and consistent vasoconstriction leading to eventual fatal right heart failure despite current medical therapies. The most common drug targets in PH are G protein coupled receptors (GPCRs), which are a target for almost a third of all FDA-approved drugs. Although these receptors have been studied intensely for over 40 years, several aspects of GPCR signaling remain poorly understood. Canonically, it has been well established that these receptors are able to signal through both heterotrimeric G proteins and β-arrestins (βarrs). These events were thought to be largely separable given that G proteins primarily initiate downstream signaling while βarrs can signal and regulate receptor desensitization and trafficking. Recent studies have suggested evidence for a combined role of G protein and βarr in GPCRs signaling through the formation of signaling “megaplexes” and the impairment of βarr-based signaling in the absence of functional G proteins. However, there remains a significant knowledge gap surrounding the significance of G protein and βarr coordinated signaling. Our long term aim is to understand the signaling mechanisms of GPCRs to provide better insight for the development of novel therapeutics for PH. In our recent studies, we have directly assessed whether G proteins and βarrs can interact across a panel receptors and were surprised to find that all receptors tested could form a complex between the inhibitory G protein (Gαi) and βarr, including the type 1 angiotensin II receptor (AT1R) and atypical chemokine receptor 3 (ACKR3, also known as CXCR7), which are both potential drug targets in PH. We further found that these complexes could interact with secondary effectors, most notably extracellular signal-regulated kinase (ERK). These results suggested a conserved, non-canonical role for Gαi:βarr signaling across GPCRs. Our overarching goal is to define the mechanism in which Gαi:βarr form complexes and understand their impact on physiology. We hypothesize that Gαi:βarr complex formation require a discrete set of motifs present in Gαi, βarrs and GPCRs and that these complexes regulate endothelial function in PH. To test this hypothesis, first I will determine the specific sequence motifs in Gαi, βarr and the receptor that are required to form Gαi:βarr complex. Second, I will determine the signalling pathways that are regulated by Gαi:βarr interaction using APEX proximity labeling and novel “complex BRET” assays. Third, I will determine the impact of Gai:βarr within PH patient endothelial cells by targeting Gαi and βarr signaling and testing their effects on endothelial function. This study strives to understand an emerging paradigm in GPCR signalling in which Gαi and βarr work together to orchestrate unique downstream signalling. Completion of these aims will provide novel insights for cell signalling, development of new pharmacological tools targeting Gαi:βarr coupling, and lay the groundwork for therapeutics for cardiovascular-related diseases. These studies will also provide an excellent opportunity for my training to develop as an independent scientist.

抽象的肺动脉高压（PH）的特点是内皮功能障碍、不规则血管重塑和尽管目前有药物治疗，但持续的血管收缩导致最终致命的右心衰竭。 PH 最常见的药物靶点是 G 蛋白偶联受体 (GPCR)，它是几乎所有药物的靶点尽管这些受体已经被深入研究了 40 多年，人们对 GPCR 信号传导的几个方面仍知之甚少。这些受体能够通过异三聚体 G 蛋白和 β-抑制蛋白 (βarrs) 发出信号。鉴于 G 蛋白主要启动下游信号传导，而 βarrs 被认为在很大程度上是可分离的可以发出信号并调节受体脱敏和贩运。最近的研究提供了证据。通过形成信号传导，G 蛋白和 βarr 在 GPCR 信号传导中发挥联合作用 “megaplexes”以及在缺乏功能性 G 蛋白的情况下基于 βarr 的信号传导受损。关于 G 蛋白和 βarr 协调的重要性仍然存在显着的知识差距我们的长期目标是了解 GPCR 的信号传导机制，以便为研究提供更好的见解。 PH 新型疗法的开发在我们最近的研究中，我们直接评估了 G 是否存在。蛋白质和 βarrs 可以跨一组受体相互作用，并且惊讶地发现所有测试的受体可以在抑制性 G 蛋白 (Gαi) 和 βarr 之间形成复合物，包括 1 型血管紧张素 II 受体（AT1R）和非典型趋化因子受体3（ACKR3，也称为CXCR7），两者都是潜在的我们进一步发现这些复合物可以与大多数次级效应物相互作用。尤其是细胞外信号调节激酶（ERK），这些结果表明它是一种保守的、非典型的。 Gαi:βarr 信号在 GPCR 中的作用我们的首要目标是定义其中的机制。 Gαi:βarr 形成复合物并了解其对生理学的影响我们突袭了 Gαi:βarr 复合物。形成需要 Gαi、βarrs 和 GPCR 中存在的一组离散基序，并且这些复合物调节为了检验这个假设，首先我将确定 Gαi、βarr 中的特定序列基序。以及形成 Gαi:βarr 复合物所需的受体其次，我将确定信号传导途径。使用 APEX 邻近标记和新颖的“复杂 BRET”测定法通过 Gαi:βarr 相互作用进行调节。第三，我将通过靶向 Gαi 和 βarr 来确定 Gai:βarr 对 PH 患者内皮细胞的影响信号传导并测试它们对内皮功能的影响。 GPCR 信号传导范例，其中 Gαi 和 βarr 共同协调独特的下游信号传导。这些目标的完成将为细胞信号传导、新药理学的开发提供新的见解。靶向 Gαi:βarr 偶联的工具，并为心血管相关疾病的治疗奠定基础。这些研究也将为我作为一名独立科学家的培训提供绝佳的机会。