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

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

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