AGS3-based optogenetic GDI induces GPCR-independent Gβγ signaling and macrophage migration

G protein-coupled receptors (GPCRs) are efficient Guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the G subunit of G protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GGTP, and free G{beta}{gamma} and are major disease drivers. Evidence shows that the ambient low threshold signaling required for cells is likely supplemented by signaling regulators such as non-GPCR GEFs and Guanine nucleotide Dissociation Inhibitors (GDIs). Activators of G protein Signaling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signaling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3s G protein regulatory (GPR) motif, to understand its GDI activity and induce standalone G{beta}{gamma} signaling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases G{beta}{gamma}, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signaling pathways and triggering GPCR-independent G{beta}{gamma} signaling in cells and in vivo.