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

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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.