Additional file 8: Figure S8. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Evolution of TM4/5 and TM5/6 interfaces as devised from MD simulations of the adenosine A1R-A2AR heterotetramer in complex with Gi and Gs. (A) Representative snapshots (20 structures collected every 25 ns) of the TM domains of A1R bound to Gi (red), Gi-unbound A1R (orange), A2AR bound to Gs (dark green), and Gs-unbound A2AR (light green). TM helices 4 and 5 are highlighted in light blue and gray, respectively. Initial (at 0 ns, transparent cylinders) and final (at 500 ns, solid cylinders) snapshots of TM interfaces are shown for homodimerization (TM4/5, within rectangles) and heterodimerization (TM5/6, within a circle) bundles. TM helices 4 (light blue), 5 (gray), and 6 (orange and green) are highlighted. (B) Root-mean-square deviations (rmsd) on protein α-carbons of the four-helix bundles forming the TM5/6 interface (orange solid line), TM4/5 interface of A1R (blue dotted line), and TM4/5 interface of A2AR (blue solid line) throughout the MD simulation. (C) Contact maps of the TM4/5 interface (rectangles in panel A) in the A1R or A2AR homodimer (left and right panels) and of the TM5/6 interface (circle in panel A) in the A1R-A2AR heterodimer (middle panel). Darker dots show more frequent contacts. (D) Detailed view of the extensive network of hydrophobic interactions (mainly of aromatic side chains) within the TM4/5 (left and right panels) and TM5/6 (middle panel) interfaces. The amino acids are numbered following the generalized numbering scheme of Ballesteros and Weinstein [37, 38]. This allows easy comparison among residues in the 7TM segments of different receptors. (TIF 4004 kb

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

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

Additional file 9: Figure S9. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Positioning YFP in the C-tail of A2AR. The complex between the A2AR protomer (in light green) and Gs (α-subunit in dark grey and yellow, β-subunit in light gray, and γ-subunit in purple) was constructed from the crystal structure of β2 in complex with Gs [33]. Although the exact conformation of the A2AR C-tail (102 amino acids, Gln311–Ser412) cannot unambiguously be determined, its orientation was modeled as in the C-tail of squid rhodopsin [39], which contains the conserved amphipathic helix 8 that runs parallel to the membrane and an additional cytoplasmic helix 9. Thus, the C-tail of A2AR expands (see solid light green line) and points intracellularly toward the N-termini of the γ-subunit as suggested for OXER [32]. The laboratory of Kostenis has shown that the C-terminal of OXER, labeled with Rluc (OXER-Rluc), gets close to the N-terminal of the γ-subunit, labeled with GFP (γ-GFP) [32]. Analogously, we propose that YFP attached to the C-tail of A2AR is positioned near the N-termini of the γ-subunit (in purple). (TIF 2395 kb

Additional file 7: Figure S7. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Molecular dynamics (MD) simulation of the adenosine A1R-A2AR heterotetramer in complex with Gi and Gs. (A) Root-mean-square deviations (rmsd) on protein α-carbons of the whole system (black solid line), of the two A1Rs (orange and red solid lines), of the two A2ARs (light and dark green solid lines), of Gi (gray solid line), and of Gs (gray dotted line) throughout the MD simulation. This color scheme matches with the color of the different proteins depicted in the two adjacent schematic representations. (B) Intermolecular distances between the N-terminal helices of the γ-subunit of Gi and Gs (magenta line), the N-terminal helices of the α-subunit of Gi and Gs (gray line), the N-terminal helix of the α-subunit of Gi and the C-terminal helix (Hx8) of inactive A1R (orange line), the N-terminal helix of the α-subunit of Gs and the C-terminal Hx8 of inactive A2AR (green line), the C-terminal Hx8 of A1R and A2AR (blue lines). These computed intermolecular distances are depicted as double arrows in the two adjacent schematic representations. (TIF 6973 kb