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

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

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

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

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

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