Identifying Novel Signaling Mechanisms Downstream of Cardiac Gq-Coupled Receptors

鉴定心脏 Gq 偶联受体下游的新型信号传导机制

• 批准号: 10535591
• 负责人: Joseph F Loomis
• 金额: $ 3.91万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535591
• 关键词: ADRBK1 gene; Adenoviruses; Adrenergic Agents; Angiotensin II; Animal Model; Apoptosis; Attenuated; Biological Assay; Biophysics; Biotin; Biotinylation; Cardiac; Cardiac Myocytes; Cardiac development; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cause of Death; Cell Surface Receptors; Cell physiology; Cells; Chromatin Remodeling Factor; Co-Immunoprecipitations; Communities; Coupled; Diglycerides; Disease; Endothelin A Receptor; Endothelin-1; Family; Functional disorder; Future; G alpha q Protein; G-Protein-Coupled Receptors; GTP-Binding Proteins; Guanosine Triphosphate; HDAC9 gene; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Heterotrimeric GTP-Binding Proteins; Human; Hydrolysis; Hyperactivity; Hypertrophy; In Vitro; Inositol; Knock-out; Label; Laboratories; Lead; Ligase; Link; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Molecular; Pathology; Phenylephrine; Phosphatidylinositols; Phospholipases A; Physiological; Play; Protein Array; Protein Isoforms; Protein Kinase C; Proteins; Proteomics; Radiolabeled; Rattus; Regulation; Research; Role; SMARCD3 gene; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Stimulus; Streptavidin; System; Testing; Therapeutic; Variant; Work; base; experimental study; follow-up; in vivo; insight; knock-down; novel; phospholipase C beta; pressure; receptor; reconstitution; response; screening; tripolyphosphate

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.

项目摘要 歧管G蛋白偶联受体（GPCR）在人心脏中表达。激活后，这些细胞 - 表面受体（包括血管紧张素II，𝛽肾上腺肾上腺素，肾上腺素和内皮素-1受体）有效 信号通路是自适应和适应不良心血管生理学的基础的。一个重要的子集 在这些心脏GPCR夫妇到GQ家族的异三聚体G蛋白中，该蛋白由𝛼，𝛽和𝛾组成 亚基。先前的研究首先将依赖G𝛼Q的信号与心脏肥大和心肌细胞联系起来 凋亡。但是，主动G𝛼Q信号促进这些病理的分子机制是 不完全理解。编纂的G𝛼Q信号通路涉及刺激磷脂酶Cβ （plc𝛽）通过G𝛼Q-GTP的同工型。 plc𝛽然后催化磷脂酰肌醇4,5-双磷酸的水解成 肌醇1,4,5-三磷酸和二酰基甘油，从而导致细胞内Ca2+信号传导和蛋白激酶 C激活。 PLC𝛽的G𝛼Q-GTP激活参与心脏肥大，但活性G𝛼Q亚基也可能 通过替代效应（包括p63rhogef和三人组）以非依赖性的方式信号。而且， 我们的实验室最近在蛋白质组学的接近性标记中发现了一系列潜在的G𝛼Q效应器 屏幕。该屏幕涉及基于细胞接近靶蛋白的基于细胞的生物素化，该蛋白是由诱饵催化的 蛋白质（野生型G𝛼Q或组成型活性G𝛼Q Q209L）与涡轮增压酶（一种混杂的生物素连接酶）融合在一起。 生物素化靶蛋白通过链霉亲和素下拉捕获，并用蛋白质组学质量鉴定 光谱法。这种方法使许多蛋白质的高信心鉴定 与含有G𝛼Q-WT-涡轮的细胞相比，有选择地富集了含有G𝛼Q-Q209L-涡轮的细胞。 这些蛋白质包括已知的G𝛼Q相互作用者（PLC𝛽，三重奏和GRK2）；但是，散布在这些已知的 相互作用者是几种蛋白质，以前尚未证明与活性G𝛼Q相互作用。这些令人兴奋 结果，加上科学界对G𝛼Q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G𝛼Q拥有AS- 参与G𝛼Q介导的心脏细胞生理和疾病的尚有刺激性作用。我会 通过两部分方法检验该假设。在AIM 1中，我将采用基于细胞的测定和体外生物物理 确定我们接近标签的初步命中次数（总计5-10个）的实验 蛋白质组学屏幕直接与主动G𝛼Q相互作用。在AIM 2中，我将测量肥大和凋亡 受假定G𝛼Q效应的siRNA敲低的心肌细胞，尤其是SmarCD3（一种调节） SWI/SNF染色质重塑复合物的成分在我的最初研究中表现出了希望。 累积地，我提出的研究将定义有助于G𝛼Q的信号通路 心肌细胞肥大，因此将指导抗击心血管疾病的治疗策略。