Schematic representation of the input forms of GPCR-SAS and the possible outcomes.

<p>The input of GPCR-SAS consists in two main sections: (i) Positions and Sequence: where the user can introduce a position/set of positions and a residue or sequence motif and (ii) Classification, that provides navigable multilevel hierarchical classification of GPCRs in families, branches and various levels of subfamilies according to different implemented schemes.</p

GPCR-SAS: A web application for statistical analyses on G protein-coupled receptors sequences - Fig 3

<p><b>Conservation analysis queries for position 3.40 (A-C) and for Ile at position 3.40 (D) in human non-olfactory class A GPCRs</b>. <b>(A)</b> the frequencies of amino acids at position 3.40; <b>(B</b>) the histogram of the amino acid frequencies at position 3.40 for the most the major subcategories (blue arrow); <b>(C)</b> the entropy of position 3.40; (<b>D</b>) the frequencies of Ile at position 3.40 for class A GPCRs compared the other classes (B, C and F; Same Level panel) and for the different class A branches (Sub-Level panel). Gray arrows indicate that it is possible to get the list of receptors that contain a certain residue or motif; the black arrow indicates that a click on the residue type in (<b>A)</b> provides the output displayed in (<b>D)</b>.</p

GPCR-SAS: A web application for statistical analyses on G protein-coupled receptors sequences

<div><p>G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. They mediate signal transduction across cell membranes and are important targets for the pharmaceutical industry. The G Protein-Coupled Receptors—Sequence Analysis and Statistics (GPCR-SAS) web application provides a set of tools to perform comparative analysis of sequence positions between receptors, based on a curated structural-informed multiple sequence alignment. The analysis tools include: (i) percentage of occurrence of an amino acid or motif and entropy at a position or range of positions, (ii) covariance of two positions, (iii) correlation between two amino acids in two positions (or two sequence motifs in two ranges of positions), and (iv) snake-plot representation for a specific receptor or for the consensus sequence of a group of selected receptors. The analysis of conservation of residues and motifs across transmembrane (TM) segments may guide the design of more selective ligands or help to rationalize activation mechanisms, among others. As an example, here we analyze the amino acids of the “transmission switch”, that initiates receptor activation following ligand binding. The tool is freely accessible at <a href="http://lmc.uab.cat/gpcrsas/" target="_blank">http://lmc.uab.cat/gpcrsas/</a>.</p></div

Additional file 2: of Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain

Table S1. List of target sequences and template structures used to construct the computer models of A1-A2AHet in complex with Gi and Gs. (PDF 23 kb

Proposed functional properties of CB₁R-5-HT_2AR heteromers.

<p>In (A), agonist binding to CB₁R (blue) or 5-HT_2AR (light green) triggers the conformational changes of TMs 5 and 6, opening the intracellular cavity for Gi and Gq binding, respectively. In (B), the formation of the CB₁R-5-HT_2AR heteromer makes both receptors signal via Gi. In (C), rimonabant binding to CB₁R or MDL 100,907 to 5-HT_2AR stabilizes the closed conformation of the receptor, facilitating heterodimerization via TMs 5 and 6 as in the crystal structure of the μ-opioid receptor. In this assembly, both protomers are locked in the closed conformation since the opening of TMs 5 and 6 for G-protein binding is not feasible (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002194#pbio.1002194.s008" target="_blank">S7 Fig</a>). Bidirectional cross antagonism is due to the fact that antagonist binding to any protomer must, in addition to its common role in a monomeric signaling unit, disrupt this very stable four-helix association. (D) In agreement, bidirectional cross antagonism is abrogated following treatment with TM 5 or TM 6 interference peptides (dark blue), which disrupt the heteromer structure.</p

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

5-HT_2AR and CB₁R form heteromers in transfected cells.

<p>In (A), BRET saturation experiments were performed in HEK-293T cells transfected with 0.025 μg of 5-HT_2AR-Rluc cDNA and increasing amounts of CB₁R-YFP cDNA (0.05 μg to 1.5 μg, black curve), with 0.5 μg of dopamine D₁R-Rluc cDNA and increasing amounts of CB₁R-YFP cDNA (0.5 μg to 6 μg, yellow line), or with 0.025 μg of 5-HT_2AR-Rluc cDNA and increasing amounts of adenosine A₁R-YFP cDNA (0.05 μg to 1.5 μg, red line). The relative amount of BRET is given as a function of 100 x the ratio between the fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc). BRET is expressed as milli BRET units (mBU) and is given as the mean ± standard deviation (SD) of 3–6 experiments grouped as a function of the amount of BRET acceptor. In (B), a schematic representation of fluorescence complementation experiments is depicted in the left panel showing that fluorescence only appears after the YFP Venus hemiprotein complementation due to the proximity of two receptors fused to hemi-YFP Venus proteins (cYFP or nYFP). In the right panel, fluorescence at 530 nm was detected in HEK-293T cells transfected with different amounts of cDNA corresponding to both 5-HT_2AR-cYFP and CB₁R-nYFP (equal amount for each construct), but not in negative controls in which cells were transfected with cDNA corresponding to 5-HT_2AR-cYFP and the noninteracting adenosine A₁ receptor-nYFP or CB₁R-nYFP and the noninteracting dopamine D₁ receptor-cYFP. One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant fluorescence over basal values in HEK-293T cells (** <i>p</i> < 0.01, *** <i>p</i> < 0.001). In (C), PLAs were performed in HEK-293T cells expressing CB₁R and 5-HT_2AR. Confocal microscopy images (superimposed sections) are shown in which heteromers appear as green spots. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μm. In (D), PLAs were performed in nontransfected HEK-293T cells, cells transiently transfected with 0.5 μg of CB₁R or 5-HT_2AR cDNA (negative controls, white columns), or with increasing amounts of CB₁R and 5-HT_2AR cDNA (black columns). In each case, the ratio between the number of green spots and the number of cells showing spots (ratio r) was calculated. One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant PLA staining over nontranfected cells (*** <i>p</i> < 0.001).</p

Interacting protomer domains in CB₁R-5-HT_2AR heteromers and heteromer disruption by TM interference peptides.

<p>In (A), HEK-293T cells expressing CB₁R and 5-HT_2AR were treated for 4 h with vehicle (left panel) or 4 μM of CB₁R TM 7, TM 5, or TM 6 interference peptides before performing proximity ligation assays. Confocal microscopy images (superimposed sections) are shown in which heteromers appear as green spots in cells treated with vehicle and with TM 7 interference peptide, but not in cells treated with TM 5 or TM 6 interference peptides. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μm. In (B–D), HEK-293T cells expressing CB₁R and 5-HT_2AR were preincubated for 20 min with rimonabant (1 μM, RIM) or MDL 100,907 (300 nM, MDL) before stimulation for 10 min (B) or 5 min (C, D) with the CB₁R agonist WIN 55,212–2 (100 nM), the 5-HT_2AR agonist DOI (100 nM), or both in the presence (B) or absence (C, D) of 0.5 μM forskolin. In (B), cAMP production was determined. Values represent mean ± SEM of <i>n</i> = 3–9 and are expressed as the percentage of the cAMP produced in forskolin-treated cells. Quantification of phosphorylated ERK 1/2 (C) or Akt (D) was determined by western blot. Values, expressed as a percentage of basal (nontreated cells), were mean ± SEM of <i>n</i> = 3–6. One-way ANOVA followed by a Bonferroni post hoc tests showed a significant effect over forskolin’s effects alone in each condition (B) or over basal (C, D) (* <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> <0.001) or of the antagonist plus agonist treatment over the agonist treatment (# <i>p</i> < 0.05, ## <i>p</i> < 0.01, ### <i>p</i> < 0.001).</p

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

Differential expression of CB₁R-5-HT_2AR heteromers in the brain detected by heteromer signaling.

<p>Slices from the hippocampus (A), caudate-putamen (B), cortex (C), and nucleus accumbens (D) of WT mice (white bars) and 5-HT_2AR KO mice (black bars) were preincubated or not with CB₁R antagonist rimonabant (1 μM, RIM) or the 5-HT_2AR antagonist MDL 100,907 (300 nM, MDL) for 20 min before the addition of the CB₁R agonist WIN 55,212–2 (1 μM, WIN), the 5-HT_2AR agonist DOI (1 μM), or both for an additional incubation period of 10 min. ERK 1/2 phosphorylation was determined by western blot. Immunoreactive bands from three to seven slices obtained from ten WT or KO animals were quantified for each condition. Values represent mean ± SEM of the percentage of phosphorylation relative to basal levels found in untreated slices. No significant differences were obtained between the basal levels of the WT and the KO mice. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (* <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001) effect over basal or of the antagonist plus agonist treatment over the agonist treatment (# <i>p</i> < 0.05, ## <i>p</i> < 0.01).</p