G-protein regulatory network governs receptor internalization dynamics.

G protein-coupled receptors (GPCRs) enable chemical communication between cells and are involved in nearly all essential functions. They transduce signals via heterotrimeric G proteins and are regulated by internalization, a process which redirects them from the cell surface to internal compartments and enables diversified signaling through spatial reorganization. Beyond the receptor, a vast regulatory network exists to further control G-protein signaling. However, it is unclear whether these modes of G-protein regulation also impact the upstream GPCR. Here, we systematically address how G-protein cycle regulation shapes GPCR internalization and establish several key principles and mechanisms governing this process. We find that timing of G-protein activation and deactivation and changes in G-protein cycle lifetime imparted by guanine nucleotide exchange factors, activators of G-protein signaling, and regulators of G-protein signaling can alter internalization outcomes. Furthermore, we determine how the activity and balance of discrete G-protein components interact with the G protein-coupled receptor kinase system to influence GPCR spatial distribution. Finally, we uncover that disease-associated variants of the most abundant G protein in the brain, GαoA, affect the regulatory network that drives GPCR internalization. Altogether, this study reveals that GPCR internalization is not a fixed receptor property but is dynamically governed by receptor-G-protein activation order, cycle lifetime, and the balance of Gα and Gβγ availability. As such, alterations in receptor internalization dynamics may contribute to the complex disease phenotypes associated with dysregulated G-protein networks.