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，不规则的血管重塑和 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。这个 在PH中最常见的药物靶点是G蛋白偶联受体(GPCRs)，它是几乎一个 FDA批准的所有药物中的第三种。尽管这些受体已经被深入研究了40多年， GPCR信号的几个方面仍然知之甚少。规范地说，已经很好地确立了 这些受体能够通过异源三聚体G蛋白和β-arrestins(β受体)来传递信号。这些事件 被认为在很大程度上是可分离的，因为G蛋白主要启动下游信号，而β是ARR 可以发出信号并调节受体的脱敏和运输。最近的研究表明，有证据表明 G蛋白和βARR通过信号形成在GPCRS信号转导中的联合作用 “巨丛”和基于βARR的信号在缺乏功能性G蛋白的情况下的损害。然而， 关于G蛋白和βARR协调的意义，仍然存在着显著的知识差距 发信号。我们的长期目标是了解GPCRs的信号机制，以便更好地了解 肺高压的新疗法的发展。在我们最近的研究中，我们直接评估了G 蛋白质和β受体可以通过一组受体相互作用，并惊讶地发现所有接受测试的受体 可以在抑制性G蛋白(GαI)和βARR之间形成复合体，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)，这两个都是潜在的 PH中的药物靶点。我们进一步发现，这些复合体可以与次级效应器相互作用，大多数 尤其是细胞外信号调节激酶(ERK)。这些结果表明了一种守恒的、非正则的 GαI的作用：跨GPCR的βARR信令。我们的首要目标是定义 GαI：βARR形成复合体并了解它们对生理的影响。我们假设GαI：βARR复合体 形成需要一组离散的基序，这些基序存在于GαI、βARR和GPCR中，这些复合体调节 高血压病患者的内皮功能。为了验证这一假设，我首先要确定GαI，βArr中的特定序列基序 以及形成GαI：βARR复合体所需的受体。其次，我将确定信令路径 它们受GαI：βARR相互作用的调控，使用APEX邻近标记和新颖的“复杂Bret”分析。 第三，我将通过靶向G-β-I和G-α-ARR来确定GAI：β-ARR对PH患者内皮细胞的影响 信号传递和测试它们对内皮功能的影响。这项研究致力于理解一种新兴的 GαI和βARR一起工作以协调独特的下行信令的GPCR信令范型。 这些目标的完成将为细胞信号转导、新药理的开发提供新的见解 针对GαI：βARR偶联的工具，为心血管相关疾病的治疗奠定了基础。 这些研究也将为我作为一名独立科学家的培训提供一个极好的机会。

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.