Identifying Novel Signaling Mechanisms Downstream of G q ‐Coupled Receptors

The G protein alpha subunit G□ q is a critical mediator of cells’ response to a variety of external stimuli as a result of activation of G q ‐coupled GPCRs. G□ q ‐dependent signaling influences an array of physiological processes, including neurotransmission and vasoconstriction; additionally, dysregulated G□ q ‐dependent signaling has been firmly linked to several pathologies, including uveal melanoma and maladaptive cardiomyocyte hypertrophy. One established mechanism by which activated G□ q subunits might promote these processes involves activation of phospholipase C beta isoforms (PLC□), which catalyze the hydrolysis of PI(4,5)P 2 into inositol (1,4,5) trisphosphate (IP 3 ) and diacylglycerol (DAG), ultimately leading to Ca 2+ mobilization and protein kinase C activation. However, G□ q subunits also signal independently of PLC□, through p63RhoGEF and Trio, for example. To identify novel G□ q targets that might be involved in G q ‐driven pathologies, we conducted a proximity biotinylation proteomic screen in HEK293A cells comparing wild‐type G□ q and constitutively active G□ q Q209L each fused to TurboID, a promiscuous biotin ligase. This approach enabled the high‐confidence identification of numerous proteins that were selectively enriched in cells expressing G□ q ‐Q209L‐TurboID compared to cells expressing G□ q ‐WT‐TurboID. These enriched proteins included known G□ q interactors (PLC□, Trio, and GRK2); however, scattered among these known interactors were several proteins that have not been previously shown to interact with active G□ q , including SPRED1, SMARCD3, YAP1, BCAS2, and HDAC9. Our initial efforts have focused on SMARCD3, a regulatory component of the SWI/SNF chromatin remodeling complex. To test for SMARCD3 interactions with G□ q , we expressed SMARCD3 in COS‐7 cells and found that SMARCD3 selectively inhibits G□ q ‐stimulated, but not G□□‐stimulated, PLC□ activity, indicating that SMARCD3 competes with PLC□ for access to active G□ q . In addition, we expressed SMARCD3 in HEK293A cells and found that (1) SMARCD3 co‐immunoprecipitates with G□ q and (2) interacts with G□ q in a nanoluciferase fragment complementation assay. SWI/SNF regulates gene transcription by modulating chromatin accessibility. Given that G q signaling regulates gene expression in both physiological and pathological systems, we will assess the impact of SMARCD3 knockdown in cellular models of G□ q ‐driven transcription and cell function. Ultimately, we will expand this approach to test other top hits' ability to interact with and potentiate signaling downstream of active G□ q . Through these endeavors, we aim to uncover novel G□ q signaling mechanisms and inform therapeutic strategies for combatting pathologies marked by aberrant G□ q signaling.