G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gαi/o signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gαi1/3-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gαi1-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gαi/o-dependent complex with a Gi-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α2A-adrenergic receptor (α2A-AR) in a Gαi/o-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α2A-AR complex occurs in the presence of Gαi/o but not Gαs or Gαq. Unexpectedly, RGS14 does not dissociate from Gαi1 in the presence of stimulated α2A-AR, suggesting preservation of RGS14·Gαi1 complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gαi1 complex and the Gαi1-dependent RGS14·α2A-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gαi/o and that these RGS14·Gαi1·GPCR complexes may be substrates for other signaling partners such as Ric-8A. Background: Regulator of G protein signaling 14 (RGS14) is a G protein regulatory (GPR) protein that participates in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Results: RGS14 forms regulated complexes with GPCRs in live cells. Conclusion: RGS14 integrates unconventional and conventional GPCR-dependent G protein signaling pathways. Significance: GPR proteins appear to be at the nexus of divergent G protein signaling pathways.