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glosensor camp reagent  (Promega)

 
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    Promega glosensor camp reagent
    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a <t>GloSensor</t> in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.
    Glosensor Camp Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/glosensor+reagent/glosensor+camp+assay/pmc12175894-268-22-26
    Average 90 stars, based on 1 article reviews
    glosensor camp reagent - by Bioz Stars, 2026-10
    90/100 stars

    Images

    1) Product Images from "Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain"

    Article Title: Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain

    Journal: Science Advances

    doi: 10.1126/sciadv.adv9267

    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a GloSensor in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.
    Figure Legend Snippet: ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a GloSensor in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.

    Techniques Used: Inhibition, Functional Assay, Activation Assay

    ( A ) Top view of the befiradol binding pose within the G s protein–bound 5-HT 1A R complex and ( B ) comparison to the binding pose in the 5-HT 1A R-G i1 protein complex. ( C ) The extended binding pocket residues F112 3.28 and M92 2.60 exhibit different conformations in the cryo-EM structure with ST171 compared to the structures with befiradol bound to 5-HT 1A R in complex with the G i and G s proteins, respectively. ( D to G ) Mutation F112 3.28 W alone and in combination with M92 2.60 F substantially decreases befiradol’s potency for the activation of G i - and G s -mediated signaling and β-arrestin 2 recruitment at the 5-HT 1A R, while the signaling profile of ST171 is not affected. For both ligands, the relative efficacies remain unchanged compared to serotonin. Data were obtained with the cAMP GloSensor measuring the inhibition of forskolin-promoted cAMP accumulation in (D) parental HEK293A and (E) HEK cells deficient of G s proteins. (F) In HEK cells deficient in G i/o proteins, an accumulation of cAMP was observed with befiradol. (G) β-Arrestin 2 recruitment was monitored by BRET in HEK293T cells with elevated GRK2 levels. Data are normalized to the maximal response of serotonin (100%) and shown with ±SEM of n = 3 to 14 independent experiments.
    Figure Legend Snippet: ( A ) Top view of the befiradol binding pose within the G s protein–bound 5-HT 1A R complex and ( B ) comparison to the binding pose in the 5-HT 1A R-G i1 protein complex. ( C ) The extended binding pocket residues F112 3.28 and M92 2.60 exhibit different conformations in the cryo-EM structure with ST171 compared to the structures with befiradol bound to 5-HT 1A R in complex with the G i and G s proteins, respectively. ( D to G ) Mutation F112 3.28 W alone and in combination with M92 2.60 F substantially decreases befiradol’s potency for the activation of G i - and G s -mediated signaling and β-arrestin 2 recruitment at the 5-HT 1A R, while the signaling profile of ST171 is not affected. For both ligands, the relative efficacies remain unchanged compared to serotonin. Data were obtained with the cAMP GloSensor measuring the inhibition of forskolin-promoted cAMP accumulation in (D) parental HEK293A and (E) HEK cells deficient of G s proteins. (F) In HEK cells deficient in G i/o proteins, an accumulation of cAMP was observed with befiradol. (G) β-Arrestin 2 recruitment was monitored by BRET in HEK293T cells with elevated GRK2 levels. Data are normalized to the maximal response of serotonin (100%) and shown with ±SEM of n = 3 to 14 independent experiments.

    Techniques Used: Binding Assay, Comparison, Cryo-EM Sample Prep, Mutagenesis, Activation Assay, Inhibition

    Related Articles

    Transfection:

    Article Title: Intrinsic molecular proton sensitivity underlies GPR4 effects on retrotrapezoid nucleus (RTN) neuronal activation and CO<sub>2</sub>-stimulated breathing
    Article Snippet: Research Articles | Cellular/Molecular Intrinsic molecular proton sensitivity underlies GPR4 effects on retrotrapezoid nucleus (RTN) neuronal activation and CO2-stimulated breathing https://doi.org/10.1523/JNEUROSCI.0799-24.2024 Received: 29 April 2024 Revised: 28 June 2024 Accepted: 30 July 2024 Gonye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.. This Early Release article has been peer reviewed and accepted, but has not been through the composition and copyediting processes.The final version may differ slightly in style or formatting and will contain links to any extended data.

    Article Title: Intrinsic Molecular Proton Sensitivity Underlies GPR4 Effects on Retrotrapezoid Nucleus Neuronal Activation and CO 2 -Stimulated Breathing
    Article Snippet: The following day, cells were transfected with the GloSensor -22F cAMP plasmid (Promega E2301) and WT or mutant GPR4 (final concentration 0.02 ng/μl); all GPR4 constructs contain a C-terminal myc-tagged mouse GPR4 expressed in pcDNA3.1 ( ). .. Constructs were mixed with Lipofectamine 2000 (Thermo Fisher Scientific 11668027) and added to cells according to manufacturer instructions and allowed to incubate for 20 h. The next day, transfection medium was removed and replaced with Hank's balanced salt solution (HBSS; Invitrogen 14175-095) containing 2% v / v GloSensor Reagent (Promega E1290). ..

    Incubation:

    Article Title: Antimuscarinic drugs exert β-arrestin-biased agonism at the muscarinic acetylcholine type 1 receptor
    Article Snippet: .. Then, cells were incubated with Glosensor reagent (Promega, Madison, WI, USA, 10 μl/well) for two hours in the dark. ..

    Article Title: Structural insight into the self-activation and G-protein coupling of P2Y2 receptor.
    Article Snippet: .. On the next day, the cells were washed once and incubated in HBSS buffer supplemented with 2% GloSensor reagent (Promega). ..

    Article Title: Structure-activity relationship analysis of meta-substituted N-cyclopropylmethyl-nornepenthones with mixed KOR/MOR activities.
    Article Snippet: Substance Use Disorder (SUD) remains a significant global challenge, with current treatment options offering limited efficacy.. Agonists targeting the kappa opioid receptor (KOR), especially those with additional mu opioid receptor (MOR) antagonistic activity, have shown promise in addressing SUD.. In this study, a series of metasubstituted N-cyclopropylmethyl-nornepenthone derivatives were designed and synthesized, and their biological activities were assessed, leading to the identification of a KOR/MOR dual modulator, compound 10a.

    Construct:

    Article Title: Intrinsic Molecular Proton Sensitivity Underlies GPR4 Effects on Retrotrapezoid Nucleus Neuronal Activation and CO 2 -Stimulated Breathing
    Article Snippet: The following day, cells were transfected with the GloSensor -22F cAMP plasmid (Promega E2301) and WT or mutant GPR4 (final concentration 0.02 ng/μl); all GPR4 constructs contain a C-terminal myc-tagged mouse GPR4 expressed in pcDNA3.1 ( ). .. Constructs were mixed with Lipofectamine 2000 (Thermo Fisher Scientific 11668027) and added to cells according to manufacturer instructions and allowed to incubate for 20 h. The next day, transfection medium was removed and replaced with Hank's balanced salt solution (HBSS; Invitrogen 14175-095) containing 2% v / v GloSensor Reagent (Promega E1290). ..

    Concentration Assay:

    Article Title: Structure-activity relationship analysis of meta-substituted N-cyclopropylmethyl-nornepenthones with mixed KOR/MOR activities.
    Article Snippet: Substance Use Disorder (SUD) remains a significant global challenge, with current treatment options offering limited efficacy.. Agonists targeting the kappa opioid receptor (KOR), especially those with additional mu opioid receptor (MOR) antagonistic activity, have shown promise in addressing SUD.. In this study, a series of metasubstituted N-cyclopropylmethyl-nornepenthone derivatives were designed and synthesized, and their biological activities were assessed, leading to the identification of a KOR/MOR dual modulator, compound 10a.



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    Promega glosensor camp reagent
    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a <t>GloSensor</t> in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.
    Glosensor Camp Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/glosensor+reagent/glosensor+camp+assay/pmc12175894-268-22-26
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    glosensor camp reagent stock solution  (Promega)
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    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a <t>GloSensor</t> in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.
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    glosensor reagent  (Promega)
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    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a <t>GloSensor</t> in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.
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    Image Search Results


    ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a GloSensor in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.

    Journal: Science Advances

    Article Title: Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain

    doi: 10.1126/sciadv.adv9267

    Figure Lengend Snippet: ( A ) An inhibition of 5-HT 1A R–mediated cAMP accumulation is observed for serotonin, befiradol, and ST171. The stimulation of cAMP accumulation in the presence of PTX suggests an additional G s response for befiradol and serotonin (serotonin: EC 50 = 52 ± 6 nM; befiradol: EC 50 = 25 ± 11 nM) but not for ST171. Data were obtained in three to five independent experiments with a CAMYEL assay in CHO-K1 cells. ( B ) Inhibition of cAMP accumulation by serotonin, befiradol, and ST171 in HEK293A cells deficient of G s proteins and ( C ) accumulation of cAMP by serotonin and befiradol but not ST171 in HEK293A cells deficient of G i/o proteins. Data were obtained with a GloSensor in n = 6 experiments, each performed in triplicates. ( D ) ST171 inhibits cAMP accumulation caused by 1 μM serotonin in HEK293AΔG i/o cells and thus demonstrates functional antagonism for the activation of G s proteins (IC 50 = 3.9 ± 0.8 nM). Data were obtained with the GloSensor in n = 3 independent experiments, each performed as triplicates. ( E ) β-Arrestin 1 and ( F ) β-arrestin 2 recruitment is monitored by bystander BRET in HEK293T cells with elevated GRK2 levels [ n = 4 to 6 (β-arrestin 1) or 11 to 14 (β-arrestin 2) independent experiments]. ( G ) ST171 is a functional antagonist (IC 50 = 3.9 ± 0.5 nM) for 5-HT 1A R–mediated β-arrestin 2 recruitment stimulated with serotonin (1 μM, ~EC 80 ). Data were obtained with the PathHunter assay in the absence of GRK2 in three independent experiments, each performed in triplicates. All data were normalized to the maximum response of serotonin and are indicated as the means ± SEM of n independent experiments.

    Article Snippet: After incubation, CP4 medium was exchanged with Hanks’ balanced salt solution [with added glucose (1 g/liter) and NaHCO 3 (0.35 g/liter)] containing GloSensor cAMP Reagent (3%, Promega) and plates were incubated for 60 min.

    Techniques: Inhibition, Functional Assay, Activation Assay

    ( A ) Top view of the befiradol binding pose within the G s protein–bound 5-HT 1A R complex and ( B ) comparison to the binding pose in the 5-HT 1A R-G i1 protein complex. ( C ) The extended binding pocket residues F112 3.28 and M92 2.60 exhibit different conformations in the cryo-EM structure with ST171 compared to the structures with befiradol bound to 5-HT 1A R in complex with the G i and G s proteins, respectively. ( D to G ) Mutation F112 3.28 W alone and in combination with M92 2.60 F substantially decreases befiradol’s potency for the activation of G i - and G s -mediated signaling and β-arrestin 2 recruitment at the 5-HT 1A R, while the signaling profile of ST171 is not affected. For both ligands, the relative efficacies remain unchanged compared to serotonin. Data were obtained with the cAMP GloSensor measuring the inhibition of forskolin-promoted cAMP accumulation in (D) parental HEK293A and (E) HEK cells deficient of G s proteins. (F) In HEK cells deficient in G i/o proteins, an accumulation of cAMP was observed with befiradol. (G) β-Arrestin 2 recruitment was monitored by BRET in HEK293T cells with elevated GRK2 levels. Data are normalized to the maximal response of serotonin (100%) and shown with ±SEM of n = 3 to 14 independent experiments.

    Journal: Science Advances

    Article Title: Discovery of a functionally selective serotonin receptor (5-HT 1A R) agonist for the treatment of pain

    doi: 10.1126/sciadv.adv9267

    Figure Lengend Snippet: ( A ) Top view of the befiradol binding pose within the G s protein–bound 5-HT 1A R complex and ( B ) comparison to the binding pose in the 5-HT 1A R-G i1 protein complex. ( C ) The extended binding pocket residues F112 3.28 and M92 2.60 exhibit different conformations in the cryo-EM structure with ST171 compared to the structures with befiradol bound to 5-HT 1A R in complex with the G i and G s proteins, respectively. ( D to G ) Mutation F112 3.28 W alone and in combination with M92 2.60 F substantially decreases befiradol’s potency for the activation of G i - and G s -mediated signaling and β-arrestin 2 recruitment at the 5-HT 1A R, while the signaling profile of ST171 is not affected. For both ligands, the relative efficacies remain unchanged compared to serotonin. Data were obtained with the cAMP GloSensor measuring the inhibition of forskolin-promoted cAMP accumulation in (D) parental HEK293A and (E) HEK cells deficient of G s proteins. (F) In HEK cells deficient in G i/o proteins, an accumulation of cAMP was observed with befiradol. (G) β-Arrestin 2 recruitment was monitored by BRET in HEK293T cells with elevated GRK2 levels. Data are normalized to the maximal response of serotonin (100%) and shown with ±SEM of n = 3 to 14 independent experiments.

    Article Snippet: After incubation, CP4 medium was exchanged with Hanks’ balanced salt solution [with added glucose (1 g/liter) and NaHCO 3 (0.35 g/liter)] containing GloSensor cAMP Reagent (3%, Promega) and plates were incubated for 60 min.

    Techniques: Binding Assay, Comparison, Cryo-EM Sample Prep, Mutagenesis, Activation Assay, Inhibition