Image_2_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

Image_3_Neuronal Calcium and cAMP Cross-Talk Mediated by Cannabinoid CB1 Receptor and EF-Hand Calcium Sensor Interactions.TIFF

<p>Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB₁ receptors (CB₁R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB₁R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca²⁺-dependent manner. Assays in neuronal primary cultures showed that, CB₁R-NCS1 complexes predominate at basal Ca²⁺ levels, whereas in the presence of ionomycin, a calcium ionophore, CB₁R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB₁R to NCS1 is required for CB₁R-mediated signaling, while the binding of CB₁R to calneuron-1 completely blocked G_i-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB₁R agonist challenge.</p

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

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

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

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

Additional file 3: Figure S3. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Controls of cAMP production and BRET assays in cells expressing minigenes and in cells expressing the ghrelin GHS1a receptor instead of one of the adenosine receptors. (A,B) cAMP determination in HEK-293T cells transfected with (A) 0.3 μg of cDNA corresponding to A1R or (B) with 0.2 μg of cDNA corresponding to A2AR with (control) or without 0.5 μg of cDNA corresponding to minigenes coding for peptides blocking either Gi or Gs binding. Cells were stimulated with the A1R agonist N6-Cyclopentyladenosine (CPA) (10 nM, red bars) in the presence of 0.5 μM forskolin (Fk) or with the A2AR agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS-21680) (200 nM, blue bars). Values expressed as % of the forskolin-treated cells (CPA reduces forskolin-induced cAMP levels, red bars) or of the basal (CGS 21680 per se enhances cAMP levels, blue bars) are given as mean ± SD (n = 4–8). One-way ANOVA followed by a Bonferroni post - hoc test showed a significant effect of CPA when compared with that of forskolin (red bars, ***p < 0.001) or of CGS 21680 when compared to basal cAMP levels (blue bars, ## p < 0.01, ### p < 0.001). (C, D) BRET saturation curves were performed in HEK-293T cells transfected with (C) 0.3 μg cDNA coding for A1R-Rluc, increasing amounts of cDNA coding for A1R-YFP (0.1–1.5 μg cDNA), and 0.4 μg cDNA coding for GHS1a, or (D) with 0.2 μg of cDNA coding for A2AR-Rluc, increasing amounts of cDNA coding for A2AR-YFP (0.1–1.0 μg cDNA), and 0.5 μg cDNA coding for to GHS1a. Prior to BRET determination, cells were treated for 16 h with medium (black curves), with 10 ng/ml of pertussis toxin (green curves), or with 100 ng/ml of cholera toxin (red curves). mili BRET units (mBU) are given as the mean ± SD (n = 4–6 different experiments grouped as a function of the amount of BRET acceptor). (TIF 1418 kb

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