12915_2016_247_MOESM3_ESM.tif (1.39 MB)

Additional file 3: Figure S3. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

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posted on 2016-04-05, 05:00 authored by Gemma Navarro, Arnau Cordomí, Monika Zelman-Femiak, Marc Brugarolas, Estefania Moreno, David Aguinaga, Laura Perez-Benito, Antoni Cortés, Vicent Casadó, Josefa Mallol, Enric Canela, Carme Lluís, Leonardo Pardo, Ana García-Sáez, Peter McCormick, Rafael Franco
Controls of cAMP production and BRET assays in cells expressing minigenes and in cells expressing the ghrelin GHS1a receptor instead of one of the adenosine receptors. (A,B) cAMP determination in HEK-293T cells transfected with (A) 0.3 μg of cDNA corresponding to A1R or (B) with 0.2 μg of cDNA corresponding to A2AR with (control) or without 0.5 μg of cDNA corresponding to minigenes coding for peptides blocking either Gi or Gs binding. Cells were stimulated with the A1R agonist N6-Cyclopentyladenosine (CPA) (10 nM, red bars) in the presence of 0.5 μM forskolin (Fk) or with the A2AR agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS-21680) (200 nM, blue bars). Values expressed as % of the forskolin-treated cells (CPA reduces forskolin-induced cAMP levels, red bars) or of the basal (CGS 21680 per se enhances cAMP levels, blue bars) are given as mean ± SD (n = 4–8). One-way ANOVA followed by a Bonferroni post - hoc test showed a significant effect of CPA when compared with that of forskolin (red bars, ***p < 0.001) or of CGS 21680 when compared to basal cAMP levels (blue bars, ## p < 0.01, ### p < 0.001). (C, D) BRET saturation curves were performed in HEK-293T cells transfected with (C) 0.3 μg cDNA coding for A1R-Rluc, increasing amounts of cDNA coding for A1R-YFP (0.1–1.5 μg cDNA), and 0.4 μg cDNA coding for GHS1a, or (D) with 0.2 μg of cDNA coding for A2AR-Rluc, increasing amounts of cDNA coding for A2AR-YFP (0.1–1.0 μg cDNA), and 0.5 μg cDNA coding for to GHS1a. Prior to BRET determination, cells were treated for 16 h with medium (black curves), with 10 ng/ml of pertussis toxin (green curves), or with 100 ng/ml of cholera toxin (red curves). mili BRET units (mBU) are given as the mean ± SD (n = 4–6 different experiments grouped as a function of the amount of BRET acceptor). (TIF 1418 kb)


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