title: 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.
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.
authors: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322. Electronic address: cvellan@emory.edu.
authors: Christopher P
authors: CP
authors: Vellano
authors: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425.
authors: Ellen M
authors: EM
authors: Maher
authors: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
authors: John R
authors: JR
authors: Hepler
authors: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425. Electronic address: blumerjb@musc.edu.
authors: Joe B
authors: JB
authors: Blumer
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D020710:RGS Proteins; C491400:RGS14 protein, human; D043562:Receptors, G-Protein-Coupled; C106374:Rgs14 protein, rat; C561938:Ric8A protein, human; C485885:Ric8a protein, rat; D019206:GTP-Binding Protein alpha Subunits, Gi-Go