Functionality of dopamine D₄ receptors in pineal gland and pinealocytes.

<p>Pineal glands extracted at 9:00 h were treated for 10 min with increasing amounts of dopamine or with 1 µM of RO 10-5824 (RO). The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A) and Akt (Ser⁴⁷³) phosphorylation (B), of two separate experiments performed in duplicate were quantified and values represent the mean ± S.D. of the fold increase relative to basal levels found in untreated cells. Significant differences with respect to basal levels were determined by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001). A representative Western blot is shown at the top (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). (C) Pinealocytes were isolated from pineal glands extracted at 9:00 h and were treated with medium (Control), 1 µM of RO 10-5824 (RO), 1 µM phenylephrine (Phenyl), or 1 µM isoproterenol (Iso) for 10 min before labeling with anti-S-arrestin (green) and anti-phospho-ERK1/2 (red), as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cell nuclei were stained with DAPI (blue). Scale bar, 5 µm.</p

D₄ receptors form heteromers with α_1B and β₁ receptors in the pineal gland.

<p>In (A to C), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and stained using anti-S-arrestin antibody (green) and anti-D₄ (A), anti-α_1B (B), or anti-β₁ (C) antibodies (red) as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Scale bar, 5 µm. In (D to F), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and the expression of α_1B-D₄ (D) and β₁-D₄ (E) receptor heteromers was visualized as punctate red fluorescent spots detected by confocal microscopy using the proximity ligation assay (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). Any expression of α_1B-β₁ receptor heteromers was seen (F). Scale bar, 20 µm. In (G and H), co-immunoprecipitation of D₄ and α_1B or D₄ and β₁ receptors from pineal gland extracted at 9:00 h (sunrise) or at 20:00 h (sunset) was performed. Glands were solubilized and processed for immunoprecipitation as described under <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a> using goat anti-D₄, rabbit anti-α₁, or goat anti-β₁ receptor antibodies or goat anti-adenosine A_2B receptor antibody as a negative control (N.C.). Solubilized gland membranes (G) and immunoprecipitates (H) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-α₁, rabbit anti-β₁ receptor antibodies, or goat anti-β₁ receptor antibody. Immunoprecipitation experiments with anti-α₁ or anti-β₁ receptor antibodies (right image in H) were performed with pineal glands extracted at 9:00 h. IP, immunoprecipitation; WB, western blotting; MW, molecular mass.</p

Functional characteristics of α_1B-D₄ and β₁-D₄ receptor heteromers in pineal gland.

<p>Pineal glands extracted at 9:00 h (A and B) or at 20:00 h (C and D) were treated for 10 min with RO 10-5824 (RO), phenylephrine (Phenyl), or isoproterenol (Iso) at 1 µM concentration alone or in combination. The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) (A and C) or Akt (Ser⁴⁷³) (B and D) phosphorylation, of three experiments performed in duplicates were quantified, and values represent the mean ± S.E.M. of the fold increase with respect to basal levels found in untreated pineal glands. Significant differences were calculated by a one-way ANOVA followed by post hoc Bonferroni's tests (**<i>p</i><0.01 and ***<i>p</i><0.001, as compared to the basal level. ^#<i>p</i><0.05 and ^##<i>p</i><0.01, as compared to the sample treated with phenylephrine; ^$<i>p</i><0.05 and ^$$$<i>p</i><0.001, as compared to the sample treated with isoproterenol). A representative Western blot is shown at the bottom of each panel.</p

Cross-antagonism between D₄ and α_1B or β₁ receptors in transfected cells and in pineal gland.

<p>In (A to D) CHO cells were transiently co-transfected with 2 µg of plasmid coding for D₄ receptors and with 3 µg of plasmid coding for α_1B receptors (A and B) or β₁ receptors (C and D). In (E and F) rat pineal glands were extracted at 9:00 h and processed as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cells were treated for 7 min and pineal glands were treated for 10 min with 500 nM of RO 10-5824 (RO), phenylephrine (Phenyl), or isoproterenol (Iso) or with 1 µM of L-745,870 (L-745), REC 15/2615 (REC), or CGP 20712 (CGP), alone or in combination. The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A, C, and E) and Akt (Ser⁴⁷³) phosphorylation (B, D, and F) of four experiments were quantified and values represent the mean ± S.E.M. of the fold increase with respect to basal levels found in untreated cells. Significant differences were calculated by a one-way ANOVA followed by post hoc Bonferroni's tests (***<i>p</i><0.001, as compared to the basal level; ^#<i>p</i><0.001, as compared to the sample treated with RO 10-5824; ^$<i>p</i><0.001, as compared to the sample treated with phenylephrine; ^&<i>p</i><0.001, as compared to the sample treated with isoproterenol). A representative Western blot is shown at the top of each panel.</p

D₄ receptors form heteromers with α_1B and β₁ receptors in transfected cells.

<p>(A) BRET saturation curves were performed in HEK-293T cells co-expressing a constant amount of D₄-RLuc construct (2 µg of plasmid transfected) and increasing amounts of β₁-YFP construct (0.4–5 µg plasmid transfected, red), α_1B-YFP construct (0.4–5 µg of plasmid transfected, blue), or D₁-YFP construct (1–4 µg of plasmid transfected, gray) or with cells co-expressing a constant amount of α_1B-RLuc construct (3 µg of plasmid transfected) and increasing amounts of β₁-YFP construct (0.4–5 µg of plasmid transfected, green). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm equal expression of Rluc construct (∼100,000 luminescence units) while monitoring the increase of YFP construct expression (2,000 to 40,000 fluorescence units). Milli BRET Units (mBU) are BRET ratio (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>)×1,000 and are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET acceptor normalized with respect to the BRET donor (YFP/RLuc). (B and C) BRET was determined in HEK-293T cells expressing a constant amount of D₄-RLuc construct (2 µg of plasmid transfected) and (B) α_1B-YFP construct (4 µg of plasmid transfected) or (C) β₁-YFP construct (4 µg of plasmid transfected) and increasing amounts (2–12 µg of plasmid transfected) of (B) α_1B receptor (red) or β₁ receptor (blue) or (C) β₁ receptor (red) or α_1B receptor (blue). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm that there were no changes in the expression of D₄-RLuc, α_1B-YFP, or β₁-YFP constructs. BRET data (see above) are expressed as means ± S.D. of three different experiments. Significant differences with respect to cells not expressing α_1B or β₁ receptors were calculated by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05 and **<i>p</i><0.01). (D) Confocal microscopy images of HEK-293T cells transfected with 1 µg of plasmid coding for D₄-RLuc and 0.5 µg of plasmid coding for α_1B-YFP or β₁-YFP. Proteins were identified by fluorescence or by immunocytochemistry. D₄-RLuc receptor is shown in red, α_1B-YFP and β₁-YFP receptors are shown in green, and co-localization is shown in yellow. Scale bar, 5 µm. (E and F) Co-immunoprecipitation of D₄ and α_1B or D₄ and β₁ receptors expressed in HEK-293T cells. Membranes from cells transfected with the indicated receptors were solubilized and processed for immunoprecipitation as described under <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a> using goat anti-D₄R, rabbit anti-α₁ or goat anti-β₁ receptor antibodies, or as negative controls (NC), goat anti-adenosine A_2B receptor antibody (top in F) or rabbit anti-adenosine A₁ receptor antibody (bottom in F). Solubilized membranes (E) and immunoprecipitates (F) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-YFP, rabbit anti-α₁, or goat anti-β₁ antibody. IP, immunoprecipitation; WB, Western blotting (numbers are included to delineate the different lanes on the SDS-PAGE); MW, molecular mass.</p

Functional characteristics of α_1B-D₄ and β₁-D₄ receptor heteromers in transfected cells.

<p>CHO cells were transfected with 2 µg of plasmid coding for D₄ receptors or with 3 µg of plasmid coding for α_1B receptors or β₁ receptors alone (A) or in combination (B to G). In (A), the selectivity of ligands was tested by measuring ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) and Akt (Ser⁴⁷³) phosphorylation in cells expressing D₄, α_1B, or β₁ receptors, treated for 7 min with 1 µM RO 10-5824, phenylephrine, or isoproterenol. In (B to E), cells expressing D₄ and α_1B receptors (B and C) or D₄ and β₁ receptors (D and E) were treated for 7 min with increasing concentrations of phenylephrine (B and C) or isoproterenol (D and E) in the presence (○) or in the absence (•) of 500 nM RO 10-5824. The immunoreactive bands, corresponding to ERK 1/2 (B and D) and Akt (C and E) phosphorylation of four experiments, were quantified and expressed as mean ± S.E.M. of arbitrary units. In (F and G) membranes of cells expressing D₄ and α_1B receptors (F) or D₄ and β₁ receptors (G) were used to perform competition binding experiments of α₁ receptor antagonist [³H]prazosin (1 nM) versus increasing concentrations of phenylephrine (1 nM to 1 mM) (F) or β₁ receptor antagonist [³H]CGP-12177 (1 nM) versus increasing concentrations of isoproterenol (1 nM to 1 mM) (G) in the presence (○) or in the absence (•) of 500 nM RO 10-5824.</p

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

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

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

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