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: 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

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Preclinical Testing of Nalfurafine as an Opioid-sparing Adjuvant that Potentiates Analgesia by the Mu Opioid Receptor-targeting Agonist Morphine

doi: 10.1124/jpet.118.255661

Figure Lengend Snippet: Nalfurafine is a potent G protein-biased KOR agonist. Comparison of G protein and β-arrestin signaling outcomes with a variety of KOR ligands (at indicated concentrations) compared with the reference standard U50,488. (A) G protein signaling was assessed with a GloSensor assay of cyclic AMP inhibition. (B) β-arrestin recruitment to the activated KOR was assessed via the Tango assay. (C and D) Results from GloSensor and Tango assays used to calculate bias factors for each compound from observed maximal efficacy (Emax or “Max”) and potency (EC50) values. ∆∆log(Max/EC50) values, with their 95% confidence intervals (CIs), are plotted in (D) for each compound to indicate relative (to U50,488) bias toward G protein signaling. All compounds were tested in triplicate alongside the reference standard U50,488. N ≥ 3 for all compounds.

Article Snippet: Quantification of G protein signaling-dependent changes in intracellular cyclic AMP (cAMP) levels was conducted using a GloSensor luciferase-based assay in HEK293T cells transiently transfected with 5 μ g of KOR expression vector DNA (3×HA-hKOR; www.cDNA.org ) and 5 μ g of pGloSensor-22F cAMP biosensor expression vector DNA (Promega, Madison, WI).

Techniques: Comparison, Inhibition