Journal: International Journal of Molecular Sciences

Article Title: Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

doi: 10.3390/ijms23094634

Figure Lengend Snippet: Effects of β-AR and a-AR ligands on cAMP production in B16F10 cells: ( A ) Representative image of cAMP measurements in real time using a GloSensor cAMP biosensor (bas, baseline). ( B ) Integrated cAMP responses computed as % of integrated forskolin response from tracings obtained in B16F10 cells stably expressing GloSensor-22F probe. Measurements were obtained in the absence or the presence of the β-AR agonist isoproterenol (ISO 1 μM), the antagonists propranolol and ICI 118,551 (PRO 10 μM, ICI 118,551 10 μM), isoproterenol plus either propranolol or ICI 118,551 (ISO 1 μM + PRO 10 μM or ICI118,551 10 μM), and the α2 agonists clonidine (CLO 1 μM) and ST91 (ST-91 1 μM). Data of three independent experiments are reported. * p < 0.05. ( C ) Concentration–response curves for the ligand-induced enhancement of cAMP production. Epinephrine (EPI), isoproterenol (ISO), norepinephrine (NE). EC 50 for ISO, EPI and NE was 4.6 nM, 45 nM and 284 nM, respectively.

Article Snippet: The luciferase-based intracellular cAMP probe GloSensor 22 F was purchased from Promega (Madison, WI, USA) and subcloned into puromycin resistance retroviral expression vector pQCXIP (Clontech, Mountain View, CA, USA).

Techniques: Stable Transfection, Expressing, Concentration Assay

Journal: Toxins

Article Title: Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

doi: 10.3390/toxins13070480

Figure Lengend Snippet: Human α-defensin-1 and -5 inhibit PT-mediated effects on cAMP signaling. PT (100 ng/mL) was pre-incubated with 12 µM α-defensin-1, α-defensin-5 or ß-defensin-1, or with the respective amount of solvent (H 2 O) for 15 min at room temperature and then added to iGIST sensor cells for 5 h at 37°C. For further control, cells were treated only with the solvent of PT. Then, inducing medium containing the luciferase substrate for the luminescent biosensor for cAMP was added. After 15 minutes of baseline measurement, cells were spiked with forskolin to activate adenylate cyclase and octreotide acetate to activate Gαi-coupled SSTR2 GPCR. Luminescence was recorded for further 60 minutes. ( a ) cAMP kinetic curves from one representative experiment are shown as mean ± SD (n = 3 from one experiment), con = cells treated only with forskolin (FSK) plus octreotide (Oct). ( b ) Bar graphs show baseline-subtracted area under the curve (AUC) from at least three independent experiments. Values are given as percent of PT-only-treated samples, mean ± SEM (n = at least nine from at least three independent experiments). Values for control samples are identical in both graphs. Results are shown in two separate graphs for better clarity. Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (left graph) or untreated controls (right graph) (** p ≤ 0.01,**** p ≤ 0.0001, ns not significant). ( c ) For control, iGIST sensor cells were treated only with FSK or with PT plus FSK in the absence of Oct to measure the maximal cAMP response in this assay. Values for control samples and samples treated with PT plus FSK and Oct are identical to values in ( a ).

Article Snippet: HEK293 cells (HEK-Gs/SSTR2_HA), ectopically expressing Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR as well as a luminescence cAMP probe (GloSensor-22F, Promega, Mannheim, Germany) [ ], were cultivated in DMEM/F12 (containing glutamine and sodium pyruvate) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and penicillin–streptomycin (1:100) (Thermo Fisher Scientific).

Techniques: Incubation, Solvent, Control, Luciferase, Comparison

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of DREADD-β2AR and corresponding protein sequence encoding for signal peptide (black), VSV-G epitope (magenta), hM3Dq ligand binding domains (blue), and β2AR signaling domains (green). Black arrow: start of the mature GPCR after post-translational cleavage of the signal peptide. b Orthogonal view of DREADD-β2AR-transfected HEK cells immunostained for the N-terminal VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Schematic of signaling pathways for functional validation of the DREADD-based chimeras. The heterotrimeric G protein consists of an α- and βγ-subunit. Below: hM3Dq is a Gα q -coupled receptor, whereas non-chimeric β2AR recruits G proteins with a Gα s subunit. DREADD-β2AR contains the β2AR signaling domains to recruit Gα s . The DREADDs rM3Ds couples to Gα s and hM4Di is associated with Gα i . d Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. e – g Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-β2AR ( e ); non-chimeric β2AR ( f ), hM3Dq, rM3Ds or hM4Di ( g ); or empty vector ( e – f ). Baseline measurements of 30 min (first 15 min not shown) followed by ligand application (gray arrow for onset) of either CNO or levalbuterol. Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-β2AR: CNO 0.1–10 µM), seven (Empty vector: CNO), four (Non-chimeric β2AR: Levalbuterol; Empty vector: Levalbuterol; hM3Dq: CNO; hM4Di: CNO), or three (rM3Ds: CNO) experimental repetitions. Source data are provided as a Source Data file. h Schematic of Gα q -coupled GPCR engaging in the mitogen-activated protein kinase (MAPK) pathway which induces transcription of a firefly luciferase reporter from a serum responsive element (SRE). i Endpoint measurement of SRE-dependent luciferase activity in HEK cells transfected with hM3Dq (blue), DREADD-β2AR (magenta), non-chimeric β2AR (green), rM3Ds (red), hM4Di (cyan), or empty vector (gray). Ligand stimulation either with 10 µM CNO (left) or 0.001 µM levalbuterol (right). Dashed line: level of vehicle control. Error bars: standard error of the mean. Two-sided one-sample T-test for comparing to a mean of 1 representing the vehicle control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.004 (hM3Dq: CNO); p = 0.03 (rM3Ds: CNO); p = 0.001 (hM4Di: CNO); p < 0.002 (DREADD-β2AR: CNO); p = 0.09 (Empty vector: CNO); p < 0.001 (Non-chimeric β2AR: Levalbuterol); p = 0.01 (Empty vector: Levalbuterol). N = three experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Sequencing, Ligand Binding Assay, Transfection, Staining, Protein-Protein interactions, Functional Assay, Biomarker Discovery, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of GPCR increasing baseline cAMP levels through constitutive activity. b Real-time measurement of cAMP-dependent luciferase activity during a 30 min baseline in HEK cells transfected with hM3Dq (blue), rM3Ds (red), hM4Di (cyan), DREADD-β2AR (magenta), or non-chimeric β2AR (green). Measure of center: Mean fold change compared to empty vector (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four experimental repetitions. Source data are provided as a Source Data file. c Graph shows average fold changes compared to empty vector control during the 30 min measurement in ( b ). Dashed line: level of empty vector control. Error bars: standard error of the mean. N = four experimental repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.81 (hM3Dq); p = 0.009 (rM3Ds); p = 0.48 (hM4Di); p = 0.01 (DREADD-β2AR); p = 0.009 (Non-chimeric β2AR). Source data are provided as a Source Data file. d Schematic of GPCR with constitutive activity impacting baseline MAPK signaling measured through an SRE reporter. e Endpoint measurement of SRE-dependent luciferase activity in transfected HEK cells. Dashed line: level of empty vector control. Error bars: standard error of the mean. N = seven (hM3Dq, DREADD-β2AR), four (rM3Ds, hM4Di), or nine (Non-chimeric β2AR) experimental repetitions. of four to nine repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p-values of individual T -tests without multiple testing correction: p < 0.001 (hM3Dq); p < 0.001 (rM3Ds); p = 0.30 (hM4Di); p < 0.001 (DREADD-β2AR); p < 0.001 (Non-chimeric β2AR). Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a , b Orthogonal view of HEK cells transfected with DREADD-GPR65 ( a ) or DREADD-GPR109A ( b ) immunostained for the VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Top: Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Baseline measurement of 30 min (first 15 min not shown) followed by CNO application (gray arrow for onset). Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-GPR65) or seven (Empty vector) experimental repetitions. Source data are provided as a Source Data file. d Endpoint measurement of serum responsive element (SRE)-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Ligand stimulation with CNO. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s . p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.009 (DREADD-GPR65); p = 0.09 (Empty vector). N = four (DREADD-GPR65) or three (Empty vector) experimental repetitions. Source data are provided as a Source Data file. e Top: Schematic of Gα i -coupled GPCR reducing cAMP levels after ligand stimulation through adenylyl cyclase (AC) inhibition. Forskolin induces cAMP synthesis through AC activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Baseline measurements followed by application of CNO or vehicle (gray arrow for onset) and forskolin (white arrow for onset). Measure of center: Mean fold change compared to vehicle (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = five experimental repetitions. Source data are provided as a Source Data file. f Schematic of competition assay between Gα i -coupled DREADD-GPR109A and Gα s -coupled A2B adenosine receptor (A2BAR). Simultaneous stimulation of Gα i through CNO and Gα s through NECA prevents cAMP-responsive element (CRE)-mediated luciferase reporter activity. g Endpoint measurement of CRE-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Simultaneous stimulation with CNO and 5 µM NECA. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.03 (DREADD-GPR109A); p = 0.34 (Empty vector). N = three (DREADD-GPR109A) or four (Empty vector) experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Transfection, Staining, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control, Inhibition, Competitive Binding Assay

Journal: Toxins

Article Title: Domperidone Inhibits Clostridium botulinum C2 Toxin and Bordetella pertussis Toxin

doi: 10.3390/toxins15070412

Figure Lengend Snippet: DOM reduces the PT-mediated effects on cAMP signaling. Cells were treated with DOM at indicated concentrations and PT (100 ng/mL) for 5 h at 37 °C or cells were left untreated (con). Inducing medium with luciferase substrate for the luminescent cAMP biosensor was added. Baseline was measured for 15 min, then cells were spiked with forskolin (FSK, activator of adenylate cyclase) and octreotide acetate (Oct, activator of SSTR2). Luminescence was recorded for 60 min. ( a ) cAMP kinetic curves of one representative experiment are shown. Values are given as mean ± SD, n ≥ 3 from one experiment. ( b ) Bar graph shows baseline-subtracted area under the curve (AUC) from at least three independent experiments. Values are given as percent of samples treated with PT only, mean ± SEM, n ≥ 6. ( c ) As the control, cells were treated with FSK or PT plus FSK without Oct to detect maximal cAMP response. Values for the control samples and samples treated with PT, FSK, and Oct are identical to values in ( b ). Significance was tested by one-way ANOVA analysis and Dunnett’s multiple comparisons test, and values refer to samples treated with PT only (left graph in ( b , c ), white bar) or con (right graph in ( b ), white bar), (**** p < 0.0001, *** p < 0.001, * p < 0.05, ns = not significant p > 0.05).

Article Snippet: The iGIST bioassay [ ] is based on HEK293 cells that ectopically express the Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR and a luminescent cAMP probe (GloSensor-22F, Promega, Madison, WI, USA).

Techniques: Luciferase, Control

Journal: bioRxiv

Article Title: In vivo photopharmacology with light-activated opioid drugs

doi: 10.1101/2023.02.02.526901

Figure Lengend Snippet: (A) Reaction scheme depicting the one-step alkylation procedure used to synthesize DEAC-OXM from commercially available oxymorphone ( 2 ) and DEAC-Br, as well as its photochemical conversion to the proposed primary reaction product “rearranged DEAC-OXM” (RE-DEAC-OXM), which likely occurs via a 1,4-Photo-Claisen rearrangement. (B) High pressure liquid chromatography (HPLC) chromatograms measured at 220 nm indicating predominant photoconversion of DEAC-OXM to RE-DEAC-OXM, which has a similar retention time, along with a much smaller amount of OXM in PBS (pH 7.2). (C) (Top) LC-MS (mass spectrometry) chromatogram of the reaction mixture shown in B. (Bottom) MS traces revealing that RE-DEAC-OXM has the same molecular weight as DEAC-OXM (531 Da). (D) Agonist dose-response curves at the MOR for DEAC-OXM, RE-DEAC-OXM, and OXM using the GloSensor™ assay of cAMP signaling in HEK293T cells. The solid line depicts the best-fit sigmoidal function used to derive the indicated EC 50 value. OXM EC 50 = 1.3 nM, DEAC-OXM EC 50 = 380 nM, RE-DEAC-OXM EC 50 = 1.3 µM. Data were normalized to the response produced by DAMGO (1 µM) and are expressed as mean ± SEM (n=5-10 wells per concentration).

Article Snippet: Then the SSF-MOR plasmid, GloSensor (Promega) 22F cAMP dependent reporter plasmid (Promega), and Lipofectamine 2000 (Invitrogen) in Opti-MEM were added.

Techniques: High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Molecular Weight, Produced, Concentration Assay

Journal: bioRxiv

Article Title: In vivo photopharmacology with light-activated opioid drugs

doi: 10.1101/2023.02.02.526901

Figure Lengend Snippet: (A) Agonist dose-response curves at the mu opioid receptor (MOR) for PhOX and OXM using the GloSensor™ assay of cAMP signaling in HEK293T cells. The solid line depicts the best-fit sigmoidal function used to derive the indicated EC 50 value. OXM EC 50 = 1.4 nM. Data were normalized to the response produced by DAMGO (1 µM) and are expressed as mean ± SEM (n=5 wells per concentration). (B) Antagonist dose-response curves at the MOR for NLX and PhNX in the presence of DAMGO (100 nM) using the GloSensor™ assay. Data are presented as in A. NLX IC 50 = 86 nM. (C) Agonist dose-response curves at the MOR for DAMGO in the absence and presence of NLX (100 nM) or PhOX (300 nM) using the GloSensor™ assay. Data are presented as in A. DAMGO EC 50 = 0.7 nM, DAMGO + PhOX EC 50 = 0.9 nM, DAMGO + NLX EC 50 = 32 nM. (D) Agonist dose-response curves at the MOR for DAMGO and PhOX using a NanoBiT-based luminescence complementation assay of β-arrestin signaling in HEK293T cells (n=4 wells per concentration). DAMGO EC 50 = 11 nM. Data were normalized to the maximal response to DAMGO (10 µM) and are expressed as the mean ± SEM.

Article Snippet: Then the SSF-MOR plasmid, GloSensor (Promega) 22F cAMP dependent reporter plasmid (Promega), and Lipofectamine 2000 (Invitrogen) in Opti-MEM were added.

Techniques: Produced, Concentration Assay