Understanding and Manipulating G Protein α Subunit and Phospholipase C Signaling Networks

我的实验室对G蛋白偶联受体(GPCRs)的信号转导感兴趣，特别关注 下游的第二信使通路及其如何驱动特定的生理学和病理生理学 细胞和组织。GPCRs信号通路是普遍存在和保守的，细胞反应是确定的 通过1)特定的GPCRs和G蛋白在细胞中表达，以及2)细胞如何解码产生的信号 通过这些感受器。我的职业生涯一直专注于发现新的细胞信号转导机制 在细胞和分子水平上超越了规范的GPCR范式，在这种范式中，单个GPCR只是 耦合到三个主要的信号转导通路，磷脂酶C激活，腺苷环化酶调节，和 Rhogef刺激，其余的由细胞来完成。在这份续签申请中，我们建议利用预付款 2)G蛋白α亚单位介导信号的蛋白质组学分析 β-肾上腺素能受体(β-AR)和磷脂酶C在细胞内的信号转导途径 心肌细胞。第一个项目是基于我们的蛋白质组筛查的惊人成功使用邻近性 标记质谱学，在识别蛋白质-蛋白质相互作用的同时维持细胞环境。我们建议 从这些屏幕中鉴定两个在染色质调节中起作用的新G蛋白靶点 通过GPCRs下游史无前例的机制进行重塑和基因表达。在……里面 此外，我们建议利用这种方法来鉴定由G蛋白介导的新的信号通路 GPCRs通过内吞和循环途径进行。GPCRs的细胞内信号转导是一种新的 该领域的新兴范例，但对细胞内G蛋白和效应器参与的了解要少得多 内部GPCR。第二个方向是跟进我们的发现，磷脂酶C在高尔基体中的活性 该装置由细胞内高尔基体定位的β1-肾上腺素能受体(β1ARs)调节。建议数 实验将验证这样的假设：细胞内的β受体调节心脏中一个独特的基因亚集。 高尔基体肌醇磷脂水解物对心肌肥大的调控作用 仪器。我们将确定哪些信号通路是由高尔基体β1AR调控的，而不是EPAC-PLCε- 我们之前已经描述过的PKD信号通路。最后，我们将探讨 β2AR下游的通路，在高尔基体反对肥大的β1AR信号。这很令人兴奋，因为 体内的β1AR信号是促肥大的，而β2AR信号是保护性的，并将揭示一种新的机制 用于保护性的β2AR信号。总体而言，这些实验将揭示新的信号机制 对针对GPCRs的治疗的影响。

My laboratory is interested signal transduction by G protein-coupled receptors (GPCRs) with a specific focus on the second messenger pathways downstream and how they drive physiology and pathophysiology in specific cells and tissues. GPCRs signaling pathways are ubiquitous and conserved, with cellular responses determined by 1) the specific GPCRs and G proteins expressed in the cell and 2) how the cells decode signals generated by these receptors. My career has been focused on uncovering novel cellular signal transduction mechanisms at a cellular and molecular level that move beyond a canonical GPCR paradigm where individual GPCRs simply couple to 3 major signal transduction pathways, phospholipase C activation, adenylyl cyclase regulation, and RhoGEF stimulation, and the cells do the rest. In this renewal application we propose to capitalize on advances made in the previous funding in two general areas 1) Proteomic analysis of G protein α subunit-mediated signal transduction pathways and; 2) intracellular signaling by β-adrenergic receptors (βARs) and phospholipase C in cardiac myocytes. The first project is based on the striking success of our proteomic screens using proximity labeling mass spectrometry that maintains cell context while identifying protein-protein interactions. We propose to characterize two novel G protein targets from these screens that play roles in regulation of chromatin remodeling and gene expression through mechanisms that would be unprecedented downstream of GPCRs. In addition, we propose to leverage this method to identification of new signal pathways mediated by G proteins as GPCRs move through the endocytic and recycling pathways. Intracellular signaling by GPCRs is a new and emerging paradigm in the field but much less is known about intracellular G protein and effector engagement by internal GPCRs. The second direction will be to follow up our finding that phospholipase C activity at the Golgi apparatus is regulated by intracellular Golgi localized β1-adrenergic receptors (β1ARs). The proposed experiments will test the hypothesis that intracellular β1ARs regulate a unique subset of genes in cardiac myocytes related to cardiac hypertrophy through regulation of phosphoinositide hydrolysis at the Golgi apparatus. We will determine what signaling pathways are regulated by Golgi β1ARs beyond the Epac-PLCε- PKD signaling pathway that we have previously described. Finally, we will investigate the mechanisms for pathways downstream of β2ARs that oppose hypertrophic β1AR signaling at the Golgi. This is exciting because β1AR signaling in vivo is pro-hypertrophic while β2AR signaling is protective and will uncover a novel mechanism for protective β2AR signaling. Overall, these experiments will reveal novel signaling mechanisms that have implications for therapies that target GPCRs.