β-arrestin recruitment facilitates a direct association with G proteins.

G protein-coupled receptors (GPCRs) are targets for almost a third of all FDA-approved drugs. GPCRs are known to signal through both heterotrimeric G proteins and β-arrestins. Traditionally these pathways were viewed as largely separable, with G proteins primarily initiating downstream signaling while β-arrestins modulate receptor trafficking and desensitization in addition to regulating their own signaling events. Recent studies suggest an integrated role of G proteins and β-arrestins in GPCR signaling, however the cellular and biochemical requirements for G protein: β-arrestin interactions remain unclear. Here we show that G proteins and β-arrestins can directly interact. Through utilization of β-arrestin-biased receptors and artificially enforced β-arrestin relocalization, we demonstrate that recruitment of β-arrestin to the plasma membrane is sufficient to interact with the G protein Gαi. Using purified proteins, we show that Gαi directly interacts with β-arrestin. In addition, we find that Gαi family members differ in their degree of association with β-arrestin, and that a large degree of this selectivity resides within the alpha helical domain of Gαi. These findings delineate the cellular and biochemical conditions that drive direct interactions between G proteins and β-arrestins and illuminate the molecular basis for how they work together to effect GPCR signaling.