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: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: a The cubic ternary complex model for a GPCR interacting with agonists (A), inverse agonists (I) and Gs alpha subunits (Gs). In this model, the receptor can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and Gs alpha subunits. The conformational equilibrium that exists between R and R* (red arrows) explains the concept of constitutive receptor activity whereby basal activity can be observed in the absence of agonists in cells overexpressing native or constitutively active mutant β 2 ARs, as a consequence of binding to Gs proteins. b The mechanical mechanism for loading the multi-well plate into the PheraStar plate reader. The 96-well plate is placed on the mechanical plate stage and then automatically taken into the plate reader with a simple motor-driven linear movement. This process has the potential to provide a linear mechanical stimulation of the cell monolayer as the plate enters the light-tight reader. The initial entry of the plate into the reader is normally sufficient to activate the receptor. In most experiments, an initial read was taken at time zero and then the plate was immediately removed for the addition of ligands before re-entering the reader. In some experiments, the plate was added slowly manually to gain an initial GloSensor TM reading of basal levels. Created with Biorender.com.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Time-course of GloSensor TM luminescence stimulated by increasing concentrations of a isoprenaline and b salbutamol in a clonal HEK293G cell line overexpressing a transfected human TS-SNAP-β 2 AR. An initial luminescence read was made at time zero. The plate was then immediately removed from the PheraStar, agonists or HBSS added and the plate was then returned to the PheraStar and measurements continued every 1 min for 60 min. Values are mean ± SEM of 5 independent experiments. In each individual experiment, triplicate determinations were made. c Concentration–response curves of peak luminescence responses obtained for formoterol, isoprenaline, salmeterol and salbutamol in HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR. Data are expressed as a percentage of the response to 1 nM isoprenaline (after normalisation of HBSS control response to zero) obtained in each individual experiment and represent mean ± SEM from five independent experiments ( n = 5). d A comparison of the time-course of GloSensor TM basal responses to HBSS addition in HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR or wild-type (WT) HEK293G cells expressing endogenous β 2 ARs at very low levels. Values are mean ± SEM from five independent experiments. e , f Data from d showing the response in each cell line at t = 0 min prior to the addition of HBSS ( e ) and at the peak of the response to HBSS ( f ). ** p = 0.0079 (Mann–Whitney U test). At t = 0, there was no significant difference between the two cell lines ( p = 0.15; Mann–Whitney U test). Outlier analysis (both ROUT and Grubs method) confirmed that there were no significant outliers in the data sets. It should be noted that in these experiments, the zero time points were taken following initial plate loading and the plate was then immediately removed for the addition of an agonist or HBSS, and the plate was then reinserted into the plate reader.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Transfection, Concentration Assay, Recombinant, Control, Comparison, Expressing, MANN-WHITNEY

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Agonist E max , and log EC 50 determined for isoprenaline, formoterol, salbutamol and salmeterol from concentration–response curves obtained by cAMP GloSensor TM in HEK293G cells expressing endogenous β 2 ARs or HEK293G cells overexpressing recombinant TS-SNAP-β 2 AR

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Concentration Assay, Expressing, Recombinant

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Impact of mechanical stimulation on basal GloSensor TM time-course responses in a clonal HEK293G cell line overexpressing recombinant TS-SNAP-β 2 AR ( a , b ) or HEK293G cells endogenously expressing β 2 ARs ( c ). In all experiments, an initial luminescence read was made at time zero after initial plate entry into the PheraStar and before any additions. The plate was then immediately removed, HBSS or ICI-118551 added, and the plate was then returned to the PheraStar. Measurements were then made at 1 min and every min for 60 min in total. In a and c HBSS or ICI-118551 (1 μM) were added, and measurements of luminescence continued every minute from time = 1 min. a , c At 15, 30 and 45 min, the motorised stage of the PheraStar removed the plate from the instrument and then immediately returned it to the plate reader for further measurements every minute. In b no additions were made before measurements were made, although the motorised stage removed the plate and returned it to the PheraStar immediately after the time = 0 initial read to be consistent with the experiments in ( a ) and ( c ). At 30 min, the motorised stage of the PheraStar removed the plate from the instrument, HBSS or 1 μM ICI-118551 was added and the plate was then immediately returned to the plate reader for further measurements every min. In a – c values are mean ± SEM from five independent experiments. In each individual experiment, triplicate determinations were made.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Recombinant, Expressing

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: a , b Time-course of the basal GloSensor TM responses in HEK293G cells expressing HiBiT-D113A-β 2 AR, HEK293G cells expressing HiBiT-β 2 AR wild-type or native HEK293G cells with endogenous-β 2 ARs obtained in the absence ( a ) and presence ( b ) of 1 μM ICI-118551. In both ( a ) and ( b ), the plate was placed in the PheraStar at t = −15 min, the plate was then removed at time zero and HBSS or ICI-118551 (1 μM) was added, and the plate immediately returned to the PheraStar. Values are mean ± SEM from seven independent experiments. c Comparison of maximal basal peak responses obtained in HEK293G cells over-expressing HiBiT-D113A-β 2 AR, HEK293G cells over-expressing HiBiT-β 2 AR wild-type or in native HEK293G cells with endogenous expression of β 2 ARs. Values are mean ± SEM from seven independent experiments. * p < 0.05 or ** p < 0.01 (one-way ANOVA with Holm-Sidak multiple comparison test). HEK293G-HiBiT-D113A-β 2 AR versus HEK293G-HiBiT-D113A-β 2 AR in the presence of 1 μM ICI-118551 (not significant, p = 0.32); HEK293G-HiBiT-D113A-β 2 AR plus ICI-118551 versus endogenous HEK293G cells plus or minus ICI-118551 (both p = 0.035); HEK293G-HiBiT-D113A-β 2 AR plus 1 μM ICI-118551 versus wild-type β 2 AR ( p = 0.009). d Cell surface expression of HiBiT-D113A-β 2 AR in HEK293G cells. Receptor expression was monitored as reconstituted nanoluciferase luminescence following the addition of 0.2% purified LgBiT and 0.2% furimazine. Values are mean ± SEM from six independent experiments. * p < 0.05 or ** p < 0.01 (ANOVA with Tukey’s multiple comparison test for matched data). P = 0.045 and 0.005 for HiBiT-β 2 AR and HiBiT-D113A-β 2 AR, respectively relative to untransfected HEK293G cells. e Specific binding of ICI-118,551-βAla-βAla-BODIPY-X-630/650 (fluorescent ICI-118551) to HEK293G cells expressing wild-type HiBiT-β 2 AR or HiBiT-D113A-β 2 AR. Total and non-specific binding was determined following the re-complementation of full-length nanoluciferase with the addition of 0.2% purified LgBiT. Non-specific binding was determined in the presence of 50 μM ICI-118551. Specific binding was determined by subtraction of non-specific binding from the total binding at each concentration of fluorescent ICI-118551. Values are mean ± SEM from five independent experiments. f Specific binding determined with 100 nM fluorescent ICI-118551 in HEK293G cells expressing wild-type HiBiT-β 2 AR or HiBiT-D113A-β 2 AR. Data taken from ( e ) showing mean ± SEM and the individual means obtained in five separate experiments. **** p < 0.0001 (paired t -test).

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Expressing, Comparison, Purification, Binding Assay, Concentration Assay

Journal: Communications Biology

Article Title: Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

doi: 10.1038/s42003-024-06128-2

Figure Lengend Snippet: Cells were transiently transfected with the wild-type (WT) β 2 AR, a double mutant (N6A and N15A) β 2 AR or a triple mutant ((N6A, N15A, N178A) β 2 AR or a pcDNA3.1 control. Each β 2 AR construct contained an N-terminal HiBiT sequence. a Comparison of the cell surface expression of the three β 2 AR constructs. Receptor expression was monitored as reconstituted nanoluciferase luminescence following the addition of 0.2% purified LgBiT and 0.25% furimazine. Values are mean ± SEM from six independent experiments. There was no significant difference between the expression levels (one-way ANOVA with Tukey’s multiple comparison tests; p = 0.923, p = 0.252 and P = 0.427 for WT versus double mutant, WT versus triple mutant, and double mutant versus triple mutant, respectively). b Comparison of maximal peak responses to 1 μM isoprenaline produced by the three β 2 AR constructs. Values are mean ± SEM from six independent experiments. There was no significant difference between the maximal responses (one-way ANOVA with Tukey’s multiple comparisons test; p = 0.844, p = 0.946 and P = 0.663 for WT versus double mutant, WT versus triple mutant and double mutant versus triple mutant, respectively). c Time-course and d peak basal GloSensor TM responses in the presence and absence of 1 μM ICI-118551. An initial luminescence read was made at time zero. The plate was then immediately removed, HBSS or ICI-118551 added and then the plate was returned to the PheraStar. Measurements were then made at 1 min and every min for 60 min in total. Values are mean ± SEM from six independent experiments. **** p < 0.0001, ** p = 0.005; * p = 0.023 (two-way ANOVA with Tukey’s multiple comparison test). 1 μM ICI-118551 had no significant effect on the basal response to the triple mutant ( p = 0.595). e Time-course and f peak basal GloSensor TM responses in the presence and absence of 1 μM ICI-118551 or 1 μM isoprenaline f in HEK293 cells transfected with the triple β 2 AR mutant or pcDNA3.1. Values are mean ± SEM from six independent experiments. **** p < 0.0001 (two-way ANOVA with Sidak’s multiple comparisons test). The peak responses to isoprenaline were not significantly different ( p = 0.380). The peak responses to HBSS were also not significantly different ( p > 0.99). In both the triple mutant ( p > 0.77) and the pcDNA3.1 control cells ( p > 0.98), the inhibition by 1 μM ICI-118551 did not reach significance.

Article Snippet: The cAMP GloSensor TM luminescence assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Techniques: Transfection, Mutagenesis, Control, Construct, Sequencing, Comparison, Expressing, Purification, Produced, Inhibition

Journal: Journal of the Endocrine Society

Article Title: Real-Time Signaling Assays Demonstrate Somatostatin Agonist Bias for Ion Channel Regulation in Somatotroph Tumor Cells

doi: 10.1210/js.2018-00115

Figure Lengend Snippet: Effect of anti-G βγ ‒binding peptide on SS analogue signaling. Cells were incubated with (A) membrane potential dye, (B) cAMP GloSensor luciferin, or (C–E) intracellular Ca 2+ dye in the presence or absence of the G protein βγ subunit peptide blocker MPS-phosducin-like protein C-terminus (MPS-Phos, 1 µM). Cells were pretreated with MPS-Phos for 15 minutes at 28°C for cAMP GloSensor experiments or for 15 minutes at 37°C for the other experiments. (A) Hyperpolarization responses after simulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). Treatment groups were not significantly different ( P = 0.5; two-tailed t test). (B) Inhibition of NKH477 (10 µM) stimulated cAMP production by SS14 (100 nM) or SOM230 (1 µM) in the absence or presence of MPS-Phos (1 µM). Response of MPS-Phos‒treated groups was not significantly different from that of nontreated groups (two-tailed t test). (C) HEK293-Sstr2A cells were incubated with the FLUO-8 am dye for 45 minutes, washed, and then incubated with the anti- βγ peptide MPS-Phos (2 µM) for 15 minutes. Cells were then treated with saturating concentrations of SS14 (100 nM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are mean ± SEM from two different experiments, three replicates per group per experiment. (D) Intracellular Ca 2+ levels after stimulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). (E) Intracellular Ca 2+ levels after stimulation with SOM230 (1 µM) or SOM230 (1 µM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are expressed as a ratio of fluorescence intensity (F 1 /F 0 ). (A, B, D, and E) Data show the mean ± SEM from three independent experiments, three replicates per group per experiment. Black arrows indicate the addition of agonists 30 seconds after the start of the experiment. -, no ligand; SOM, SOM230; SS, somatostatin 14.

Article Snippet: Stable GH12C1 cell lines expressing a cAMP biosensor were created by transfecting the 22F GloSensor cAMP biosensor (Promega) into GH12C1-HA3-rSstr2A clone #35 cells using FuGENE (Promega) and selecting with 200 μg/mL of hygromycin B. Clonal cell lines were isolated by limiting dilution and were screened for biosensor activity.

Techniques: Incubation, Membrane, Two Tailed Test, Inhibition, Fluorescence