β 2 ar  (Bioss)

Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: In all HEK293 lines, calcium signals were recorded following a two-step addition protocol. This is exemplified in a for the β 2 AR. At t = 20 s, either solvent ( a i ) or Gq stimulus ATP 100 µM ( a ii – a iv ) was added, followed by a second addition at t = 140 s of either Iso or Calcium ionophore A23187. a iv Cells were pretreated with 1 µM of the Gq inhibitor FR. b – d Concentration-effect curves derived from the maximum calcium response of the second addition of b Iso on β 2 AR, c PGE 1 on prostanoid EP 2 and EP 4 , d NECA on A 2A and A 2B receptors, or A23187 (5 µM) after prior addition of solvent (no priming), ATP (100 µM) or CCh (100 µM). To exclude the contribution of endogenous Gi/o-coupled prostanoid and adenosine receptors to Gs-Ca 2+ , cells were pretreated overnight (16 h) with 100 ng/ml of the Gi/o inhibitor pertussis toxin (PTX). Representative traces are means + SEM, averaged data are mean ± SEM of n biologically independent experiments ( b : CCh and solvent n = 3, ATP n = 7; c : n = 3; d : n = 3), each performed in duplicate. Source data are provided as a Source Data file.

Article Snippet: The pSNAP-β 2 AR plasmid was obtained from Cisbio.

Techniques: Solvent, Concentration Assay, Derivative Assay

Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: a , b Representative Ca 2+ recordings in response to Iso addition after t = 20 s and corresponding quantification of maximum Ca 2+ peak responses collected in HEK293 ( a ) or HEK-∆Gs cells ( b ) transfected with the expression plasmid coding for the β 2 AR. HEK-∆Gs cells were cotransfected with either empty vector DNA ( b i , v ), or plasmids coding for Gα s ( b ii , iii ), or Gα q proteins ( b vi , vii ), respectively. c i Cartoon representation of the BRET-based Gq-CASE biosensor which reports separation of Gα q from Gβγ after activation as a decrease of BRET . c ii–iv Concentration-dependent activation of Gq protein BRET evoked in HEK293 cells with exogenous expression of the β 2 AR and the Gq-CASE biosensor, displayed as real-time BRET recordings and concentration-effect curve derived from the BRET changes after 9 min. d i Schematic for the BRET-based Gβγ release assay monitoring freed Gβγ dimers after G protein activation of heterotrimers harboring unmodified Gα subunits. d ii–iii Iso-induced BRET increase between Venus-labeled Gβγ and the membrane-associated C-terminal fragment of the G protein-coupled receptor kinase 3 fused to NanoLuciferase (masGRK3ct-NanoLuc), shown as real-time BRET recordings and their bar chart quantification. e Inositol monophosphate (IP 1 ) accumulation measured in naive HEK293 cells transfected to express the β 2 AR. Where indicated, cells were pretreated with FR to silence the function of Gq proteins (1 µM in a – d ; 10 µM in e ). Representative Ca 2+ traces and real-time BRET recordings are mean + SEM, averaged data are mean ± SEM of n biologically independent experiments ( a iii : n = 3; b iv : n = 3; b viii : n = 3; c iv : w/o n = 4, FR n = 5; d iii : n = 3), each performed in duplicate. IP 1 accumulation data ( e ) are mean ± SEM of four independent experiments performed in technical triplicates. Statistical significance was calculated with a two-way ANOVA with Fisher´s post-hoc analysis. Source data are provided as a Source Data file. c i and d i , created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ”.

Article Snippet: The pSNAP-β 2 AR plasmid was obtained from Cisbio.

Techniques: Transfection, Expressing, Plasmid Preparation, Activation Assay, Concentration Assay, Derivative Assay, Release Assay, Labeling, Membrane

Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: a Representative Iso-induced Ca 2+ traces and their quantification in the absence or presence of 50 µM of the IP 3 R antagonist 2-APB in naive HEK293 cells after ATP priming. b Exemplary label-free whole cell activation profiles, based on detection of dynamic mass redistribution (DMR) in response to ATP-stimulated Gq-coupled P2Y receptors in untreated (w/o), 2-APB-treated (50 µM), and FR-treated (1 µM) HEK293 cells, and corresponding quantification. c BRET-based real-time IP 3 detection following a two consecutive addition protocol. Cartoon illustrating the IP 3 intramolecular BRET biosensor principle . In IP 3 -free conditions, energy donor Renilla luciferase (Rluc) and energy acceptor Venus, each fused to the IP 3 R ligand binding domain (LBD) are in close proximity (high BRET state). Binding of an IP 3 molecule triggers donor:acceptor separation, resulting in a BRET decrease (low BRET state). BRET ratios are plotted as reciprocals of the I/I o values. d i–iii Agonist-induced IP 1 accumulation in HEK293 cells with and without ATP (100 µM) or CCh (100 µM) priming using Iso ( d i ), PGE 1 ( d ii ), and NECA ( d iii ) to stimulate β 2 AR, EP 2 /EP 4 , and A 2A /A 2B , respectively. e Iso-induced PIP 2 depletion after Gq priming. Schematic of the PIP 2 hydrolysis NanoBiT-based biosensor. PIP 2 hydrolysis is reflected by rapid translocation of the Small BiT (SmBiT)-tagged PH domain of PLCδ1 from plasma membrane-localized Large BiT (LgBiT)-CAAX to the cytosol resulting in decreased luminescence. Real-time recordings in ( a , b ) are mean values + SEM. IP 3 ( c ) and PIP 2 ( e ) recordings, concentration-effect curves ( a , b ), and bar charts ( c – e ) are mean values ± SEM for n independent biological experiments ( a : n = 4; b : n = 3; c : n = 5; d : solvent and ATP n = 4, CCh n = 3; e : n = 4). Ca 2+ measurements are duplicates; DMR, IP 1 accumulation, and PIP 2 depletion are triplicate, and IP 3 -BRET time-courses are quadruplicate determinations. Statistical significance was calculated with a two-way ANOVA with Dunnett’s ( c ) and Šídák’s ( d , e ) post-hoc analysis. Source data are provided as a Source Data file. c and e was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ”.

Article Snippet: The pSNAP-β 2 AR plasmid was obtained from Cisbio.

Techniques: Activation Assay, Luciferase, Ligand Binding Assay, Binding Assay, Translocation Assay, Membrane, Concentration Assay, Solvent

Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: a , b Representative Ca 2+ traces and corresponding quantification of maximum Ca 2+ amplitudes in HEK293 ( a ) and HEK-ΔGs ( b ) cells transfected with either empty vector DNA or cDNA plasmids coding for PLCβ3-wt, PLCβ3 ΔXY or PLCβ3 F715A upon Iso stimulation. Rightmost panels: Western blot quantification of each PLCβ variant. The statistical significance of expression level differences was determined using a one-way ANOVA with Tukey´s post-hoc analysis. c , d Naive HEK293 cells were transfected to express either PLCβ3 ΔXY ( c ) or PLCβ3 F715A ( d ) in the absence (vector) or presence of the Gβγ scavenger masGRK3ct. e Iso-induced PIP 2 depletion in HEK-ΔGq/11/12/13 cells transfected to express the PIP 2 hydrolysis NanoBiT-based biosensor along with PLCβ F715A , β 2 AR, and masGRK3ct or empty vector DNA as control. f IP 3 BRET recordings and corresponding quantification in HEK-ΔGq/11/12/13 cells, transfected to express the IP 3 -BRET sensor along with PLCβ F715A in the absence (empty vector) or presence of masGRK3ct upon addition of Iso or buffer. g Cartoon representation depicting the cellular consequences of cAMP production as well as IP 3 formation on mobilization of cytosolic Ca 2+ from ER sources. cAMP and IP 3 synergize to sensitize ER-localized IP 3 R channels for mobilization of cytosolic Ca 2+ . Mutant PLCβ3 variants with crippled autoinhibition produce IP 3 without Gq priming in response to Gβγ only. PLCβ mut = PLCβ 3 ΔXY , or PLCβ 3 F715A . Cells in a – d were FR-pretreated (1 µM) to exclude any potential Gq contribution. Representative Ca 2+ recordings in a – d are shown as mean + SEM, summarized data are mean ± SEM of n independent biological replicates ( a , b : n = 3; c , d : n = 4), each performed in duplicate. PIP 2 depletion data ( e ) are mean + SEM of n = 3 experiments, each performed in duplicate. BRET IP 3 real-time recordings ( f ) are depicted as mean + SEM of n = 3 experiments, one performed in triplicate and two in nonuplicate; their summarized data are shown as mean ± SEM; statistical significance was determined using a two-tailed student’s t test. Source data are provided as a Source Data file. g was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ”.

Article Snippet: The pSNAP-β 2 AR plasmid was obtained from Cisbio.

Techniques: Transfection, Plasmid Preparation, Western Blot, Variant Assay, Expressing, Control, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: Competition binding studies for isoprenaline, adrenaline, noradrenaline, formoterol, salbutamol, salmeterol, BI-167-107 binding Lumi4-Tb labelled DDM-TS-SNAP-β 2 AR, using 75nM CA200693 (S)-propranolol-green. A) Equilibrium measurements were read at 20 min post DDM-TS-SNAP-β 2 AR addition for all compounds except BI-167-107 which was read at 40 min. TR-FRET between Lumi4-Tb and CA200693 (S)-propranolol-green PHERAstar FSX using two laser flashes per cycle and 520/620 TRF module. Data points show mean of three experiments normalised to 0% inhibition of specific CA200693 (S)-propranolol-green bound for each compound, ± SEM. B) Comparison of IC 50 1min / IC 50 equilibrium values for all seven compounds binding DDM-TS-SNAP-β 2 AR using TR-FRET, bars show mean of three experiments ± SEM.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Binding Assay, Inhibition, Comparison

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: A summary of pK i and IC 50 1min / IC 50 equilibrium values for isoprenaline, adrenaline, noradrenaline, formoterol, salbutamol, salmeterol, and BI-167-107 binding DDM-TS-SNAP-β 2 AR obtained from equilibrium competition binding. TR-FRET between Lumi4-Tb and CA200693 (S)-propranolol-green PHERAstar FSX using 2 laser flashes per cycle and 520/620 TRF module. Values are mean of three experiments ± SEM.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: CASE G s activation studies , summarised in A) for B) Formoterol, C) Salbutamol, D) Salmeterol, E) Isoprenaline, F) Adrenaline, G) Noradrenaline, and H) BI-167-107 were fitted to the operational model. Formoterol was used as the reference ligand and K A values fixed to experimentally obtained K i values. CASE G s activation was obtained in a T-REx TM -293 -SNAP-β 2 AR and CASE G s single colony population which had been induced with 1μg/mL tetracycline for 48h. Duplicate wells of adherent cells were stimulated with varying concentrations of ligand and BRET was read at 15min post ligand addition using 550LP/450-80nm luminescence module and PHERAstar FSX, Data points show mean of duplicate wells from a representative single experiment, error bars show SD.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: 1μM purified Venus-mini-G s was incubated with DDM-β 2 AR-nLuc and varying concentrations of isoprenaline, salbutamol, formoterol, salmeterol, adrenaline, noradrenaline, or BI-167-107 at a final concentration 1% DMSO for 90min. NanoBRET between TS-SNAP-β 2 AR-nLuc and Venus-mini-G s was read on the PHERAstar FSX, at room temperature, using LUM 550LP/450-80nm module. All curves show combined normalised mean data ± SEM of three independent observations.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Purification, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: Investigation of the association and dissociation at 20min using 33μM mini-G s, of Venus-mini-G s binding to DDM-TS-SNAP-β 2 AR when preincubated with saturating concentration of B) Formoterol C) Salmeterol D) BI-167-107 E) Isoprenaline F) adrenaline G) Noradrenaline H) Salbutamol at room temperature, using LUM 550LP/450-80nm module. All figures show specific binding, where 30μM mini-G s was used to define the NSB, representative raw data of n=3, fitted to a two-phase association and one phase dissociation.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: Specific saturation binding of increasing concentrations of purified Venus-mini-G s binding to DDM-TS-SNAP-β 2 AR-nLuc in the presence of saturating concentrations of formoterol, salbutamol, salmeterol BI-167-107, isoprenaline, adrenaline and noradrenaline. nanoBRET between TS-SNAP-β 2 AR-nLuc and Venus-mini-Gs was read on PHERAstar FSX, at room temperature, using LUM 550LP/450-80nm module at 20min. Data was fitted to one-site specific binding model, points show the mean ± SEM of 4 independent experiments.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Binding Assay, Purification

Journal: bioRxiv

Article Title: Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics

doi: 10.1101/2024.01.05.574357

Figure Lengend Snippet: A) agonists of higher efficacy induce a conformation of β 2 AR that is more likely to recruit a mini-Gs protein but once bound there is no difference in the β 2 AR conformation within the agonist-β 2 AR-mini-Gs complex, B) Use of the cubic ternary complex model to investigate the effect of increasing the rate of G protein recruitment on the potency of the agonist-receptor complex to activate the G protein. Arrow indicates increases in apparent ligand EC 50 values for the formation of GαGTP. C) Use of the cubic ternary complex model to investigate the effect of increasing the rate of G protein recruitment on agonist affinity for the GPCR. Dotted line indicates log( K d ) of ligand-receptor occupancy. As shown in the figure the association rate of Ga to the receptor does not affect ligand binding affinity, hence the yellow and blue curves lie directly on top of each other.

Article Snippet: Slide-a-Lyzer dialysis cassettes, NuPAGE LDS sample buffer, NuPAGE 4-12% Bis-Tris 15 x 1.0mm well gels, NuPAGE MOPs SDS running buffer, Pageruler pre-stained protein ladder and BODIPY F-L-cystine dye were all obtained from Thermofisher (MA, U.S.A). β 2 AR antagonist [(S)-propranolol-green] (CA200693), was obtained from CellAura, UK, and supplied by Hello Bio, (Bristol, U.K) (s)-(-)- Propranolol hydrochloride, salmeterol were obtained from Tocris, (Bristol, U.K).

Techniques: Ligand Binding Assay

Journal: Circulation research

Article Title: Divergent actions of Myofibroblast and Myocyte β 2 -Adrenoceptor in Heart Failure and Fibrotic Remodeling

doi: 10.1161/CIRCRESAHA.122.321816

Figure Lengend Snippet: A ) Quantification of body weights during six months HFD in β1 2 AR-flox and β 2 AR-cKO and fKO transgenic mice. Plasma glucose levels were measured after a 6-hour fast followed by intraperitoneal injection of glucose (1g/kg), and cardiac function was measured by echocardiography, including EF, E/A ratio, and IVRT. B) Picrosirius-red staining for collagen in heart tissue was imaged and quantified; heart weights were normalized to tibia lengths. C ) RNAseq analysis of β 2 ARflox, cKO and fKO mouse hearts after NC and HFD feeding. Principal Component Analysis (PCA) and affected reactome pathways are summarized (NCBI #PRJNA891331). The circle size and color correspond to the FDR in reactome pathway analysis. D ) After MI surgery, hematoxylin and eosin and picrosirius-red staining of heart collagen was imaged and quantified; cardiac EF was measured by echocardiography in β 2 ARflox, cKO and fKO mice. E ) Immunofluorescent and western analysis of phospho- and total smad2, smad3, ERK, RSK, and cfos, and CTGF and α-SMA in overnight serum starved WT and β 2 AR-KO MEFs following stimulation with TGFβ (10 ng/ml) for indicated times and in NC- and HFD-fed β 2 AR-flox and fKO mouse hearts in vivo . Images are representative of mean quantified data. Dot-plots show mean ± SEM; and the numbers of independent samples are indicated. For fibrosis data in Figure 1B , p values were obtained by Mann Whitney test. All other datasets passed the Shapiro-Wilk normality test. p values were obtained after repeated measures AVNOA (time courses), two-way ( Figure 1A , dot plots), or one-way ( Figure 1D , dot plots) ANOVA followed by Tukey’s test.

Article Snippet: The methods, data, and materials are available upon request. β 2 AR-flox, β 2 AR-cKO (MHC-cre, JAX, 009074), β 2 AR-fKO (Postn-cre, Conway SJ at Indiana University) male mice (6–8-week-old) were randomly assigned to 6-month HFD or NC (D12492 and D12450B, Research Diets) feeding as females are resistant to HFD-induced cardiomyopathy.

Techniques: Transgenic Assay, Clinical Proteomics, Injection, Staining, RNA sequencing, Western Blot, In Vivo, MANN-WHITNEY