Identification of Gβ5 Interactome Associated with Neuronal Development in Neuro-2a Cells through miniTurbo Proximity Labeling

Abstract Gβ5 is a member of the Gβ family; however, unlike other heterotrimeric Gβ proteins, which transduce extracellular signals in response to G-protein coupled receptor (GPCR) activation, Gβ5 negatively regulates GPCR signaling by stabilizing the GTPase-activating proteins (GAP) R7 subfamily of Regulator of G-protein Signaling (R7-RGS) proteins. Mutations in Gβ5 have been implicated in a multisystem developmental disorder. While the regulation of GPCR signaling at the plasma membrane is likely important in this process, Gβ5/R7-RGS also localizes to the cytosol and nucleus. Potential protein–protein interactions of Gβ5 in these subcellular compartments are not well studied, so we sought to characterize the interactome of Gβ5 utilizing a live-cell biotin proximity-labeling assay in Neuro-2a cells coupled with mass spectrometry to identify novel protein interactions of the complex. We identified 48 potential interactors of Gβ5 with two analysis methods, most of which are previously unreported interactions important in neuronal development and function. The enriched phenotypes associated with the Gβ5 interactome in our results align with previously reported phenotypes of Gβ5 deficiencies.