(837.12) Identifying Novel Signaling Mechanisms Downstream of Gq-Coupled Receptors

Poster Board Number: B71

Joseph Loomis (University of Michigan), Naincy Chandan (University of Michigan Medical School), Matthew Brody (University of Michigan Medical School), Alan Smrcka (University of Michigan Medical School)

The G protein alpha subunit G𝛼q is a critical mediator of cells’ response to a variety of external stimuli as a result of activation of Gq-coupled GPCRs. G𝛼q-dependent signaling influences an array of physiological processes, including neurotransmission and vasoconstriction; additionally, dysregulated G𝛼q-dependent signaling has been firmly linked to several pathologies, including uveal melanoma and maladaptive cardiomyocyte hypertrophy. One established mechanism by which activated G𝛼q subunits might promote these processes involves activation of phospholipase C beta isoforms (PLC𝛽), which catalyze the hydrolysis of PI(4,5)P2 into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG), ultimately leading to Ca2+ mobilization and protein kinase C activation. However, G𝛼q subunits also signal independently of PLC𝛽, through p63RhoGEF and Trio, for example.

To identify novel G𝛼q targets that might be involved in Gq-driven pathologies, we conducted a proximity biotinylation proteomic screen in HEK293A cells comparing wild-type G𝛼q and constitutively active G𝛼q 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 G𝛼q-Q209L-TurboID compared to cells expressing G𝛼q-WT-TurboID. These enriched 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, 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 G𝛼q, we expressed SMARCD3 in COS-7 cells and found that SMARCD3 selectively inhibits G𝛼q-stimulated, but not G𝛽𝛾-stimulated, PLC𝛽 activity, indicating that SMARCD3 competes with PLC𝛽 for access to active G𝛼q. In addition, we expressed SMARCD3 in HEK293A cells and found that (1) SMARCD3 co-immunoprecipitates with G𝛼q and (2) interacts with G𝛼q in a nanoluciferase fragment complementation assay.

SWI/SNF regulates gene transcription by modulating chromatin accessibility. Given that Gq signaling regulates gene expression in both physiological and pathological systems, we will assess the impact of SMARCD3 knockdown in cellular models of G𝛼q-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 G𝛼q. Through these endeavors, we aim to uncover novel G𝛼q signaling mechanisms and inform therapeutic strategies for combatting pathologies marked by aberrant G𝛼q signaling.

Support or Funding Information

This work was supported by NIH grant R35GM127303 (to AVS).