Identifying Novel Signaling Mechanisms Downstream of Cardiac Gq-Coupled Receptors

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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.