title: Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex.
abstract: Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics (W.G.R., S.S.O., S.M.L., J.B.B.) and Department of Neurosciences (J.B.B.), Medical University of South Carolina, Charleston, South Carolina.
authors.forename: William G
authors.initials: WG
authors.lastname: Robichaux
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics (W.G.R., S.S.O., S.M.L., J.B.B.) and Department of Neurosciences (J.B.B.), Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Sukru S
authors.initials: SS
authors.lastname: Oner
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics (W.G.R., S.S.O., S.M.L., J.B.B.) and Department of Neurosciences (J.B.B.), Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Lanier
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics (W.G.R., S.S.O., S.M.L., J.B.B.) and Department of Neurosciences (J.B.B.), Medical University of South Carolina, Charleston, South Carolina blumerjb@musc.edu.
authors.forename: Joe B
authors.initials: JB
authors.lastname: Blumer
mesh_terms: D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009154:Mutation; D037342:Pertussis Toxin / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; C450520:seven-transmembrane G-protein-coupled receptor; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat