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

Christopher P Vellano,Ellen M Maher,John R Hepler,Joe B Blumer

doi:10.1074/jbc.m111.274928

Christopher P Vellano, Ellen M Maher + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.274928

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Abstract

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 Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-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 Gα(i/o)-dependent complex with a G(i)-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 α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/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·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.

Highlights

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)
Given that RGS14 is an regulators of G protein signaling (RGS) protein that interacts with G␣i/o-GTP but contains a GPR motif that binds G␣i1/3-GDP, we examined whether the RGS141⁄7G␣i1 complex can be regulated by a G␣i/o-linked GPCR
We show that RGS14 forms a stable complex with G␣i1 via its GPR motif and that this complex is proximal to GPCRs as evidenced by the presence of specific bioluminescence resonance energy transfer (BRET) signals between RGS14 and the ␣2A-adrenergic receptor (␣2A-AR) in the presence of G␣i1

Summary

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). We show that RGS14 forms a stable complex with G␣i1 via its GPR motif and that this complex is proximal to GPCRs as evidenced by the presence of specific bioluminescence resonance energy transfer (BRET) signals between RGS14 and the ␣2A-adrenergic receptor (␣2A-AR) in the presence of G␣i1 This RGS141⁄7␣2A-AR complex partially dissociates/rearranges after receptor agonist treatment and is further regulated by Ric-8A. Together, these findings illustrate that RGS14 functions together in both conventional and unconventional G protein signaling and that Ric-8A may recognize and act on GPCR1⁄7G␣i1⁄7GPR complexes to further regulate G␣i signaling

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