Identifying Novel Signaling Mechanisms Downstream of Cardiac Gq-Coupled Receptors

项目摘要 多种G蛋白偶联受体（GPCR）在人类心脏中表达。一旦激活，这些细胞- 包括血管紧张素II、β-肾上腺素能、β-肾上腺素能和内皮素-1受体在内表面受体启动 信号通路的基础上的适应性和适应不良的心血管生理。重要子集 这些心脏GPCR的偶联到Gq家族的异源三聚体G蛋白，其由α，β和β组成， 亚单位。先前的研究已经将G蛋白依赖性信号传导与心脏肥大和心肌细胞凋亡紧密联系在一起。 凋亡然而，活跃的Gβ q信号传导促进这些病理的分子机制是 没有完全理解。编码的Gβ q信号通路涉及磷脂酶C β的刺激 (PLC𝛽G-2 q-GTP的异构体。然后PLC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基甘油，从而导致细胞内Ca 2+信号传导和蛋白激酶 C激活。PLCβ的Gβq-GTP激活参与心肌肥厚，但活性Gβ q亚单位也可能 通过包括p63 RhoGEF和Trio在内的替代效应物以PLC信号转导非依赖性方式进行信号转导。此外，委员会认为， 我们的实验室最近在近距离标记蛋白质组学中发现了一种新的潜在的Gβq效应子阵列， 屏幕该筛选涉及靶蛋白的基于细胞内邻近的生物素化，其由诱饵催化 融合至TurboID（一种混杂的生物素连接酶）的蛋白质（野生型Gβ q或组成型活性Gβq Q209 L）。 通过链霉亲和素pulldown捕获生物素化的靶蛋白，并通过蛋白质组学质量鉴定 光谱法这种方法能够高置信度地鉴定出许多蛋白质， 与含有G209 q-WT-TurboID的细胞相比，在含有G209 q-Q209 L-TurboID的细胞中选择性富集。 这些蛋白质包括已知的G蛋白 q相互作用物（PLCβ、Trio和GRK 2）;然而，这些已知的相互作用物中分散着一些蛋白质。 相互作用物是先前未显示与活性Gβ q相互作用的几种蛋白质。这些令人兴奋 结果，结合科学界对G蛋白介导的心肌细胞的分散理解， 病理生理学，导致我提出的工作的中心假设。我假设格伦克拥有- 尚未表征的效应物，其参与Gdaq介导的心脏细胞生理学和疾病。我会 用两部分的方法来检验这个假设。在目标1中，我将采用基于细胞的测定和体外生物物理学方法， 实验，以确定是否有选择数量的初步命中（5-10总数），从我们的接近标记 蛋白质组筛选直接与活性G蛋白相互作用。在目标2中，我将测量 心肌细胞受到siRNA敲低假定的Gβ q效应子，特别是SMARCD 3，一种调节 SWI/SNF染色质重塑复合物的组成部分，在我的初步研究中显示出希望。 累积起来，我提出的研究将确定G蛋白依赖的信号通路，有助于 心肌细胞肥大，并因此将指导用于对抗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.