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Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex.
Robichaux, William G; Oner, Sukru S; Lanier, Stephen M; Blumer, Joe B.
Afiliação
  • Robichaux WG; 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.
  • Oner SS; 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.
  • Lanier SM; 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.
  • Blumer JB; 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.
Mol Pharmacol ; 88(2): 231-7, 2015 Aug.
Article em En | MEDLINE | ID: mdl-25972449
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 Gßγ 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 Gαßγ 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/oßγ 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 Gßγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oßγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαßγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas de Ligação ao GTP / Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP / Toxina Pertussis / Receptores Acoplados a Proteínas G Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: Mol Pharmacol Ano de publicação: 2015 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas de Ligação ao GTP / Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP / Toxina Pertussis / Receptores Acoplados a Proteínas G Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Revista: Mol Pharmacol Ano de publicação: 2015 Tipo de documento: Article