Identifying Novel Signaling Mechanisms Downstream of Cardiac Gq-Coupled Receptors

项目摘要 人类心脏表达多种G蛋白偶联受体(GPCRs)。一旦激活，这些细胞- 表面受体--包括血管紧张素II、𝛽肾上腺素能、𝛼肾上腺素能和内皮素-1受体--启动 作为适应性和非适应性心血管生理学基础的信号通路。一个重要的子集 在这些心脏GPCRs中，GQ家族的异三聚体G蛋白与𝛼，𝛽和𝛾偶联 亚单位。以前的研究已经明确地将G-𝛼Q依赖信号与心肌肥大和心肌细胞联系在一起 细胞凋亡。然而，活性G𝛼Q信号促进这些病理的分子机制是 没有完全理解。编码的G-𝛼Q信号通路涉及磷脂酶C-β的刺激 (PLC𝛽)亚型：G-𝛼Q-gtp。然后，PLC𝛽催化磷脂酰肌醇4，5-二磷酸水解成 肌醇1，4，5-三磷酸和二酰甘油，从而导致细胞内钙信号和蛋白激酶 C激活。G-𝛼Q-GTP激活PLC𝛽参与心肌肥厚，但G-𝛼Q亚基激活也可能参与心肌肥厚 通过替代效应器，包括p63Rhogef和Trio，以与𝛽无关的方式发送信号。此外， 我们实验室最近在邻近标记蛋白质组中发现了一组新的潜在G𝛼Q效应子 屏幕上。这一筛选涉及由诱饵催化的基于细胞内邻近的目标蛋白的生物素化。 蛋白质(野生型G𝛼Q或结构性活性G𝛼Q Q209L)与TurboID融合，TurboID是一种混杂的生物素连接酶。 通过链霉亲和素下拉捕获生物素化的目的蛋白，并与蛋白质组质量进行鉴定 光谱分析。这种方法能够高置信度地鉴定大量的蛋白质， 与含有G𝛼Q-WT-TurboID的细胞相比，选择性富集于含有G𝛼Q-Q209L-TurboID的细胞。 这些蛋白包括已知的G𝛼Q相互作用子(PLC𝛽、TRIO和GRK2)；然而，分散在这些已知的 相互作用物是一些以前没有被证明与活性G𝛼Q相互作用的蛋白质。这些令人兴奋的 结果：结合科学界对G𝛼Q介导的心肌细胞的零散认识 病理生理学，引出了我提议的工作的中心假设。我假设G𝛼Q具有- 参与G-𝛼-Q介导的心肌细胞生理学和疾病的尚不明确的效应因子。这就做 用两部分的方法来检验这一假设。在目标1中，我将使用基于细胞的分析和体外生物物理 实验以确定从我们的邻近标记中选择的初步命中数(总共5-10个) 蛋白质组筛选直接与活性G𝛼Q相互作用。在目标2，我将测量肥大和细胞凋亡 心肌细胞受G𝛼Q效应子的小干扰RNA敲除，特别是调控因子SMARCD3 SWI/SNF染色质重塑复合体的组成部分，在我的初步研究中显示出了希望。 总而言之，我提出的研究将定义G𝛼Q依赖的信号通路，这些信号通路有助于 心肌细胞肥大，因此，将指导抗击心血管疾病的治疗策略。

Project Summary Manifold G protein-coupled receptors (GPCRs) are expressed in the human heart. Upon activation, these cell- surface receptors—which include angiotensin II, 𝛽 adrenergic, 𝛼 adrenergic and endothelin-1 receptors—initiate signaling pathways that underlie both adaptive and maladaptive cardiovascular physiology. A significant subset of these cardiac GPCRs couple to heterotrimeric G proteins of the Gq family, which are composed of 𝛼, 𝛽, and 𝛾 subunits. Previous studies have firmly linked G𝛼q-dependent signaling to cardiac hypertrophy and cardiomyocyte apoptosis. However, the molecular mechanisms by which active G𝛼q signaling promotes these pathologies are not fully understood. The codified G𝛼q signaling pathway involves the stimulation of phospholipase C beta (PLC𝛽) isoforms by G𝛼q-GTP. PLC𝛽 then catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate and diacylglycerol, thereby leading to intracellular Ca2+ signaling and protein kinase C activation. G𝛼q-GTP activation of PLC𝛽 is involved in cardiac hypertrophy, but active G𝛼q subunits may also signal in a PLC𝛽-independent manner through alternative effectors, including p63RhoGEF and Trio. Moreover, our laboratory recently discovered a novel array of potential G𝛼q effectors in a proximity labeling proteomic screen. This screen involved in-cell proximity-based biotinylation of target proteins, which was catalyzed by bait proteins (wild-type G𝛼q or constitutively active G𝛼q Q209L) fused to TurboID, a promiscuous biotin ligase. Biotinylated target proteins were captured via streptavidin pulldown and identified with proteomic mass spectrometry. This approach enabled the high-confidence identification of numerous proteins that were selectively enriched in cells containing G𝛼q-Q209L-TurboID compared to cells containing G𝛼q-WT-TurboID. These proteins included known G𝛼q interactors (PLC𝛽, Trio, and GRK2); however, scattered among these known interactors were several proteins that have not been previously shown to interact with active G𝛼q. These exciting results, combined with the scientific community's scattered understanding of G𝛼q-mediated cardiomyocyte pathophysiology, lead to the central hypothesis of my proposed work. I hypothesize that G𝛼q possesses as- yet-uncharacterized effectors that participate in G𝛼q-mediated cardiac cell physiology and disease. I will test this hypothesis with a two-part approach. In Aim 1, I will employ cell-based assays and in vitro biophysical experiments to ascertain whether a select number of preliminary hits (5-10 total) from our proximity labeling proteomic screen directly interact with active G𝛼q. In Aim 2, I will measure hypertrophy and apoptosis in cardiomyocytes subjected to siRNA knockdown of putative G𝛼q effectors, especially SMARCD3, a regulatory component of the SWI/SNF chromatin remodeling complex that has shown promise in my initial studies. Cumulatively, my proposed research will define G𝛼q-dependent signaling pathways that contribute to cardiomyocyte hypertrophy and, thus, will guide therapeutic strategies for combatting cardiovascular disease.