Defining the Role of of Noncanonical GPCR Signalling in Pulmonary Hypertension

摘要 肺动脉高压（PH）的特征是内皮功能障碍、不规则血管重构和血管内皮细胞损伤。 持续的血管收缩导致最终致命的右心衰竭，尽管目前的药物治疗。的 PH中最常见的药物靶点是G蛋白偶联受体（GPCR），它是几乎所有PH的靶点。 三分之一的FDA批准的药物。尽管这些受体已经被深入研究了40多年， GPCR信号传导的几个方面仍然知之甚少。从规范上讲， 这些受体能够通过异源三聚体G蛋白和β-抑制蛋白（βarrs）两者发出信号。这些事件 考虑到G蛋白主要启动下游信号传导，而βarrs主要启动下游信号传导， 可以发出信号并调节受体脱敏和运输。最近的研究表明 对于G蛋白和β-内酰胺酶在GPCR信号传导中通过形成信号传导的组合作用， “megaplexes”和功能性G蛋白缺失时基于β arr的信号传导受损。然而，在这方面， 围绕G蛋白和β-内酰胺酶协调的重要性， 信号我们的长期目标是了解GPCR的信号传导机制，以便更好地了解 在我们最近的研究中，我们直接评估了G 蛋白质和βarrs可以通过a组受体相互作用， 抑制性G蛋白（Gαi）与β-D-半乳糖苷酶（β-D-半乳糖苷酶），包括1型血管紧张素II（Ang II）， 受体（AT 1 R）和非典型趋化因子受体3（ACKR 3，也称为CXCR 7），它们都是潜在的 我们进一步发现这些复合物可以与次级效应物相互作用， 特别是细胞外信号调节激酶（ERK）。这些结果表明，一个保守的，非典型的 Gαi的作用：GPCR中的β 1信号传导。我们的首要目标是定义一种机制， Gαi：β I形成复合物并了解其对生理学的影响。我们假设Gαi：β I复合物 形成需要存在于Gαi、βarrs和GPCR中的一组离散的基序，并且这些复合物调节 为了验证这一假设，首先我将确定Gαi、β i和β i中的特定序列基序， 以及形成Gαi：β I复合物所需的受体。第二，我将确定信号通路 使用APEX邻近标记和新的“复合BRET”测定法， 第三，我将通过靶向Gαi和β i来确定Gai：β i在PH患者内皮细胞内的影响。 信号传导并测试它们对内皮功能的影响。本研究旨在了解一种新兴的 在GPCR信号传导的范例中，Gαi和β i共同作用以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：β I偶联的工具，并为心血管相关疾病的治疗奠定基础。 这些研究也将为我的培训提供一个极好的机会，使我成为一名独立的科学家。

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.