Notes: HEK293 cells stably expressing the GloSensor™-20F cAMP Plasmid were used to study the molecular pharmacology of the A_2BAR -selective antagonist PSB 603. The HEK293 cells endogenously express the human A_2BAR receptor and the GloSensor™ system allowed for real-time kinetic analysis of receptor activation by reporting the levels of cellular cAMP following compound treatment. The authors were able to measure both the extent of receptor activation and also the kinetic profile of cAMP levels in the cell and concluded that PSB 603 acts as a negative allosteric modulator of A_2BAR -mediated increases in cAMP accumulation. (4970)

Notes: CHO cells expressing the human MOR were stably transfected with the GloSensor™ -22F cAMP Vector using the ViaFect™ Transfection Reagent. Inhibition of the forskolin-stimulated intracellular cAMP accumulation in the cells through the addition of the GloSensor™ cAMP Reagent (4% vol/vol). Assays were performed in 384-well plates with 5,000 cells/well in 30µl. (4668)

Notes: The authors were interested in developing a high-throughput screening method for detecting G_i-coupled receptors agonists but without the need for a cAMP inducer. HEK 293 cells were stably transfected using either pGloSensor™-22F cAMP Plasmid or pGloSensor™-22F cAMP Plasmid and a plasmid carrying an internal ribosome entry site (IRES) and succinate receptor 1 (SUCNR1) with puromycin resistance. HEK 293 cells stably carrying the pGloSensor™ plasmids were starved for 5 hours, detached from a T75 flask and incubated in the dark at room temperature with HBSS assay buffer containing 300μM IBMX and GloSensor™ cAMP Reagent. Cells were dispensed at 150,000 cells per well in 96-well plates or 37,000 cells per well in 384-well plates with various concentrations of SUCNR1 ligands. After 10 minutes, a basal level of luminescence was measured before forskolin or isoproterenol was injected (40 readings taken following injection). In some experiments, the cells were incubated 100ng/ml pertussis toxin the day prior to adding ligands or agonists. The assay system was then tested to measure the EC₅₀ value for succinic acid and compare to published literature. (4576)

Notes: The authors were interested in testing selective agonists of the κ-opioid receptor (KOR)-dynorphin with a bias for G proteins. Human embryonic kidney cells were transfected with a 1:1 ratio of a plasmid encoding KOR and the pGloSensor™-22F cAMP Plasmid. The transfected cells were plated into a 384-well plate the next day and treated with test compound for 20–30 minutes. Then GloSensor™ cAMP Reagent and isoproterenol were added, incubated for 10 minutes and G_ai-mediated activity was assessed by measuring luminescence. (4520)

Notes: These authors developed luciferase-based biosensors to monitor target cell-specific apoptosis in real time. They used GloSensor™  technology to develop biosensors containing a circularly permuted luciferase linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites.  Cleavage by the respective proteases causes activation of bioluminescence, providing a convenient way to study patterns of caspase activation by different cells of the immune system. (4527)

Notes: The authors sought to determine whether different residues had specific roles in binding and activation of the k-opioid receptor (KOR) by agonist molecules with distinct chemotypes. For function assays they introduced point mutations into human KOR using site-directed mutagenesis. All mutations were verified by Sanger automated sequencing. HEK293T cells were cotransfected with pGloSensor™-22F cAMP Plasmid plus the different KOR variants. Cells were plated, and after 24 hours, medium replaced with drug buffer and the different drug treatments. cAMP production was detected by treatment with isoproterenol in GloSensor™ Reagent. (4519)

Notes:

This paper describes use of GloSenso™ technology to develop a luciferase biosensor for detection of caspase 3/7 activity. The authors stably expressed a caspase-3/7 biosensor (pGloSensor™-30F DEVDG Vector, available as a custom product from Promega) in 1833 breast cells and D54 glioma cells, which were then used to monitor caspase activation in cell culture following addition of the cell-permeable substrate (GloSensor™ cAMP Reagent). The paper demonstrates use of this  luciferase biosensor to detect apoptosis in vivo in cultured cells and in a mouse model, and also demonstrates the utility of the technology for high-throughput screening for pharmacologically active compounds. The regulation of apoptosis by caspases is used as an example in this study, biosensors to study other proteases involved in the regulation of cellular processes can be designed using the concepts described in the paper. (4525)

Notes: These authors generated luciferase biosensors and used them to screen for potential inhibitors of Middle East respiratory syndrome coronavirus (MERS-CoV) replication. The biosensors were developed using the pGloSensor™-30F plasmid backbone and were engineered to include cleavage sites for the viral papain-like protease (PLpro) or 3-chymotrypsin-like protease (3CLpro). Lytic, endpoint assays and live-cell assays were performed to detect protease activity. The authors identified a small-molecule inhibitor that blocked the activity of MERS-CoV 3CLpro. These biosensor-based assays allowed rapid evaluation of viral protease activity and screening for protease inhibitors. (4526)

Notes: These authors characterized a novel sulindac derivative (SBA) that inhibits cell proliferation and induces apoptosis in human colon tumor cells. To investigate the effect of SBA  on intracellular cGMP levels, they transfected HEK293 cells with a GloSensor™ construct containing firefly luciferase fused to the human PDE5 GAF-A cGMP binding domain (GloSensor cGMP-40F plasmid). Binding of cGMP causes a conformational change that results in increased luminescence. Kinetic measurements of luminescence showed that intracellular cGMP levels increased upon treatment with SBA. (4524)

Notes: Sleep deprivation increases susceptibility to diseases such as diabetes and cancer. Because chronic inflammation is a feature of these diseases, the authors of this paper investigated whether the circadian oscillator component Cryptochrome (CRY) has a role in regulating the immune response. They found constitutive NF-κB and protein kinase A (PKA) activation in Cry1(-/-);Cry2(-/-) knockout mice, and high basal levels of cAMP. As part of the study, the effect of CRY overexpression on intracellular cAMP levels was evaluated in 293T cells using the bioluminescence-based GloSensor™ cAMP Assay. Overexpression of CRY1 reduced the cAMP production induced by forskolin, prostaglandin E2 or isoproterenol. Immunoprecipitation analysis with Flag-tagged CRY1 showed that CRY1 binds to adenylyl cyclase and limits cAMP production. The authors suggest that reduced CRY expression in chronic sleep deprivation may result in elevated cAMP levels, leading to increased PKA and NF-κB activation and offering a potential link between sleep deprivation, circadian rhythm disruption and chronic inflammation. (4224)

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta₂-adrenergic receptor (β₂AR ), a G_s-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β₂AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β₂AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β₂AR with isoproterenol. Endogenous β₂ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (E_max) after isoproterenol pretreatment. (4138)

Notes: The authors used stably transfected HEK cells expressing the human β2 adrenergic receptor, (β2AR) where the C-terminal tail was replaced with the C-terminal tail of the V2 vasopressin receptor (to increase signal-to-noise ratio) followed by a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline-controlled transcription factor (tTA). A second construct encoding β-arrestin 2 fused to TEV protease was also transfected into the same cell line. To follow the recruitment of β–arrestin upon ligand stimulation of the β2AR, the authors detected the cleavage of tTA and its translocation to the nucleus where it transcribed a stably expressing luciferase reporter gene. The Bright-Glo™ Luciferase Assay Reagent was used to detect luciferase activity and confirm positive recruitment of β-arrestin. The luminescent GloSensor™ cAMP Assay was used to assess ligand stimulation of the β2AR signaling through G proteins, resulting in an increase in cAMP. The authors used both of these assays to quantify ligand bias and identify weakly biased compounds of the β2AR and angiotensin II type 1A receptors. A number of known compounds were assessed to show the value of this strategy in helping to decipher complex signaling pathways. This approach may be useful in the development of novel biased ligands for therapeutic use. (4146)

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gα_q or Gα_i/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the G_i-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC₅₀ values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC₅₀ for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gα_i subunit binding responsible for enhanced GTPase activity.
(4148)