An optogenetic GDI drives GPCR and Gα independent macrophage migration

Abstract

Activators of G protein signaling (AGS) proteins play diverse functional roles in eukaryotic cells, including asymmetric cell division and membrane protein trafficking. The group II AGS (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) consisting of four G‐protein regulatory (GPR) GoLoco motifs that bind to Gαi/o in the G protein heterotrimer. AGS3 binding should therefore release Gβγ from the heterotrimer. However, the ability of AGS3 to directly signal through Gβγ in a living cell has yet to be demonstrated. To investigate the role of AGS3 in Gβγ signaling independent of G protein‐coupled receptors, we engineered optogenetic AGS3 using one or more consensus GPR sequences. This consensus sequence has shown higher binding efficacy towards Gαi GDP comparative to the wild type. Upon expression, our Opto‐AGS3 showed localized PIP3 generation and directional migration in RAW 264.7 cells due to Gβγ generation, indicating non‐canonical Gβγ signaling of AGS3. Our results also provide GPCR and Gα independent optogenetic control of Gβγ signaling with subcellular spatial and millisecond temporal control.