Cocaine binding to σ₁ receptor modulates the ERK 1/2 signaling in transfected cells.

<p>CHO cells were transfected with D₂ receptor cDNA (1 µg, black bars) or cotransfected (white bars) with D₂ receptor cDNA and σ₁ receptor siRNA (6.25 µg of oligonucleotides). Cells were incubated for 30 min (a) or 10 min (b) with medium (basal) or with 30 µM cocaine (a) or 1 µM quinpirole (b) in the absence or in the presence of 10 µM raclopride or 100 nM PD144418. In (<b>c</b>) cells were treated with medium (basal), 30 µM cocaine for 30 min, 1 µM quinpirole for 10 min or 30 µM cocaine for 30 min and, during the last 10 min, with 1 µM quinpirole. In all cases, ERK 1/2 phosphorylation is represented as percentage over basal levels (100%). Results are mean ± SEM of six to eight independent experiments performed in duplicate. Bifactorial ANOVA showed a significant (**p<0.01 and ***P<0.005) effect over basal.</p

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

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

Negative cross-talk between cocaine and the D₂ receptor agonist quinpirole on ERK 1/2 phosphorylation in mice striatum.

<p>In (<b>a</b>) WT (black bars) and σ₁ receptor KO (white bars) mouse striatal slices were treated with 1 µM quinpirole for 10 min, with 150 µM cocaine for 30 min or with cocaine for 30 min and, during the last 10 min, with quinpirole. Immunoreactive bands from six slices obtained from five WT or five KO animals were quantified for each condition. Values represent mean ± SEM of 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 σ₁ receptor KO mice. Bifactorial ANOVA showed a significant (*p<0.05, **p<0.01, ***p<0.005) effect over basal. One-way ANOVA followed by Bonferroni post hoc tests showed a significant cocaine-mediated counteraction of quinpirole (^&p<0.05, ^&&p<0.01). In (<b>b</b>) a representative scheme summarizing the overall results is shown. Top images represent D₂ and D₁ receptors signaling in the indirect and direct striatal pathway neurons after dopamine binding. Bottom images represent the effect of cocaine increasing the dopamine by inhibiting dopamine transporters (DAT) and interacting with σ₁ receptors within σ₁-D₂ and σ₁-D₁ receptor heteromers, changing the dopamine receptor signaling.</p

Molecular interaction between σ₁ receptors and D₂ receptors in living cells.

<p>BRET saturation experiments were performed with HEK-293T cells co-transfected with: (<b>a</b>) D₂-RLuc cDNA (0.4 µg, squares) or adenosine A_2A-RLuc cDNA as negative control (0.2 µg, triangles) and increasing amounts of σ₁-YFP cDNA (0.1 to 1 µg cDNA), (<b>b</b>) D₃-RLuc cDNA (0.5 µg, squares) or D₄-RLuc cDNA (0.5 µg, triangles) and increasing amounts of σ₁-YFP cDNA (0.1 to 1 µg cDNA). The relative amount of BRET acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells only expressing the donor, and the luciferase activity of the donor (YFP/Rluc). BRET data are expressed as means ± S.D. of five to six different experiments grouped as a function of the amount of BRET acceptor. In (<b>c</b>) confocal microscopy images of HEK-293T cells transfected with D₂-YFP or σ₁-RLuc (top panels) or co-transfected with D₂-YFP and σ₁-RLuc (bottom panels), treated (right images) or not (left images) with 30 µM cocaine for 30 min. σ₁ receptors (red) were identified by immunocytochemistry and D₂ receptors (green) were identified by its own fluorescence. Co-localization is shown in yellow. Scale bar:10 µm.</p

Higher order complex formation between σ₁ receptors and dopamine D₂ receptors in living cells.

<p>In (<b>a</b>) BRET saturation experiments were performed with HEK-293T cells co-transfected with σ₁-RLuc cDNA (0.2 µg) and increasing amounts of σ₁-YFP cDNA (0.1 to 0.6 µg cDNA). A schematic representation of a BRET process is shown at top in which the receptor fused to RLuc acts as donor and the receptor fused to YFP acts as acceptor. In (<b>b</b>) and (<b>c</b>) SRET saturation experiments were performed with HEK-293T cells co-transfected with: (b) a constant amount of D₂-RLuc (0.6 µg) and D₂-GFP² (1 µg) receptor cDNA (squares) or A_2A-RLuc (0.3 µg) and A_2A-GFP² (0.5 µg) receptor cDNA, as negative control (triangles), and increasing amounts of σ₁-YFP receptor (0.2 to 1.5 µg cDNA), (c) a constant amount of σ₁-Rluc (0.3 µg) and D₂-GFP² (1 µg) (triangles) or A₂-GFP² (0.5 µM) as negative control (squares) receptor cDNA and increasing amounts of σ₁-YFP receptor cDNA (0.2 to 1.5 µg). The relative amount of acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells only expressing the donor, and the luciferase activity of the donor (YFP/Rluc). A schematic representation of a SRET process is shown at top images in which two sequential energy transfer events between Rluc and GFP² (BRET process) and between GFP² and YFP (FRET process) occurs. In (<b>d</b>) BRET with luminescence/fluorescence complementation approach was performed measuring BRET in cells co-transfected with 1 µg of the two cDNAs corresponding to D₂-nRLuc8 and D₂-cRLuc8 and with 1.5 µg of the two cDNAs corresponding to σ₁-nVenus and σ₁-cVenus (5). As negative controls, cells transfected with the same amount of cDNA corresponding to D₂-nRLuc8, D₂-cRLuc8, σ₁-nVenus and cVenus (1), D₂-nRLuc8, D₂-cRLuc8, σ₁-cVenus and nVenus (2), D₂-nRLuc8, σ₁-nVenus, σ₁-cVenus and cRLuc8 (3), or D₂-cRLuc8, σ₁-nVenus, σ₁-cVenus and nRLuc8 (4) did not display any significant luminescence or positive BRET. A schematic representation of a BRET with luminescence/fluorescence complementation approach is given at the top image in which one receptor fused to the N-terminal fragment (nRluc8) and another receptor fused to the C-terminal fragment (cRluc8) of the Rluc8 act as BRET donor after Rluc8 reconstitution by a close receptor-receptor interaction and one receptor fused to an YFP Venus N-terminal fragment (nVenus) and another receptor fused to the YFP Venus C-terminal fragment (cVenus), act as BRET acceptor after YFP Venus reconstitution by a close receptor-receptor interaction. BRET or SRET data are expressed as means ± S.D. of five to six different experiments grouped as a function of the amount of BRET or SRET acceptor.</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

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

Effect of σ₁ receptor ligands on σ₁-D₂ receptor heteromer.

<p>BRET was measured in HEK-293T cells cotransfected with: (<b>a</b>) D₂–Rluc cDNA (0.4 µg) and increasing amounts of σ₁-YFP receptor cDNA (0.1 to 1 µg), (<b>b</b>) σ₁–Rluc cDNA (0.2 µg) and increasing amounts of σ₁-YFP receptor cDNA (0.1 to 1 µg), (<b>c</b>) D₂–Rluc cDNA (0.4 µg) and increasing amounts of D₂-YFP receptor cDNA (0.2 to 2 µg) or (<b>d</b>) siRNA corresponding to σ₁ receptor (see Methods), D₂–Rluc cDNA (0.4 µg) and increasing amounts of D₂-YFP receptor cDNA (0.2 to 2 µg)<b>,</b> not treated (black), treated for 30 min with 30 µM cocaine (red), treated for 10 min with 100 nM PRE084 (blue) or 1 µM PD144418 (green) or treated for 30 min with 30 µM cocaine and 1 µM PD144418 (orange)<b>.</b> The relative amount of BRET acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells only expressing the donor, and the luciferase activity of the donor (YFP/Rluc). BRET data are expressed as means ± SD of four to six different experiments grouped as a function of the amount of BRET acceptor.</p