Gtpγs

Manufactured by PerkinElmer

Sourced in United States

GTPγS is a non-hydrolyzable GTP analog that can be used to study G-protein-coupled receptor (GPCR) signaling. It binds to and activates G-proteins, allowing for the investigation of GPCR-mediated cellular responses.

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9 protocols using gtpγs

GTPγS Binding Assay for Receptor Activation

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GTPγS binding assays
were performed as described previously.^{24 (link)} Briefly, 7.5 μg of membranes were incubated for 60 min at
30 °C in a total volume of 200 μL of GTPγS binding
assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM
EGTA, and 100 mM NaCl) with unlabeled CP55,940 (at least nine different
concentrations were used ranging between 100 pM and 100 μM),
0.1 nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life
Sciences, Boston, MA), 10 μM GDP (Sigma, St. Louis, MO), and
0.1% (w/v) BSA in the absence and presence of varying concentrations
of the allosteric compounds as indicated. Nonspecific binding was
determined with 10 μM unlabeled GTPγS (Sigma, St. Louis,
MO). The reaction was terminated by rapid filtration through Whatman
GF/C filters. The radioactivity trapped in the filters was determined
by liquid scintillation counting.

Khurana L., Ali H.I., Olszewska T., Ahn K.H., Damaraju A., Kendall D.A, & Lu D. (2014). Optimization of Chemical Functionalities of Indole-2-carboxamides To Improve Allosteric Parameters for the Cannabinoid Receptor 1 (CB1). Journal of Medicinal Chemistry, 57(7), 3040-3052.

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Veratridine and NBQX Administration Protocol

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Veratridine and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) were purchased from Tocris Bioscience (UK). Veratridine (50 μM) [31 (link),83 (link)] and NBQX (100 μM) [84 (link),85 (link)], were infused through the intracerebral probe into the DRN of the animals.
[³³P]α-dATP (2′ deoxyadenosine 5′-(α-thio) triphosphate at a specific activity of >2500 Ci/mmol was purchased from Perkin Elmer and GTPγS (10 μM) and used for the mTOR and BDNF in situ hybridization studies.

Garro-Martínez E., Fullana M.N., Florensa-Zanuy E., Senserrich J., Paz V., Ruiz-Bronchal E., Adell A., Castro E., Díaz Á., Pazos Á., Bortolozzi A, & Pilar-Cuéllar F. (2021). mTOR Knockdown in the Infralimbic Cortex Evokes A Depressive-like State in Mouse. International Journal of Molecular Sciences, 22(16), 8671.

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GTPγS Binding Assay for 2-AG

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GTPγS
binding assays were performed as described previously.^{62 (link)} Briefly, 6 μg of membranes was incubated
for 60 min at 30 °C in GTPγS binding assay buffer (50 mM
Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl)
with the unlabeled 2-arachidonoylglycerol (2-AG) (at least nine different
concentrations were used ranging between 100 pM and 1 μM), 0.1
nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences,
Boston, MA), 10 μM GDP (Sigma, St. Louis, MO), and 0.1% (w/v)
BSA in the absence and presence of 10 μM test compounds. The
effect of the compound on inhibiting the level of basal GTPγS
binding was evaluated in the absence of agonist. Nonspecific binding
was determined with 10 μM unlabeled GTPγS (Sigma, St.
Louis, MO). After rapid filtration through Whatman GF/C filters the
radioactivity trapped in the filters was determined by liquid scintillation
counting.

Scott C.E., Ahn K.H., Graf S.T., Goddard WA I.I.I., Kendall D.A, & Abrol R. (2015). Computational Prediction and Biochemical Analyses of New Inverse Agonists for the CB1 Receptor. Journal of Chemical Information and Modeling, 56(1), 201-212.

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Evaluating CB1 Receptor G-Protein Coupling

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To evaluate the impact of the test compounds on the G-protein coupling efficiency of the CB₁ receptor, GTPγS assays were performed as described previously.^{30 (link)} Briefly, 7.5 μg of membrane preparation expressing the CB₁ receptor was incubated with a saturating concentration of CP55,940 (1 μM), 0.1 nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), 5 μM GDP (Sigma, St. Louis, MO), and 0.1% (w/v) BSA in the absence and presence of varying concentrations of the test allosteric modulators. GTPγS binding assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl) was used to make a total volume of 200 μL, and the membranes were incubated at 30 °C for an hour. Nonspecific binding was determined with 10 μM unlabeled GTPγS (Sigma, St. Louis, MO). For the controls, membrane preparations expressing CB₁ receptor were treated with DMSO or a high concentration of the inverse agonist SR141716A (1 μM). Membrane preparations from the same cells not transfected with CB₁ were also evaluated for the G-protein coupling levels to determine non-CB₁ mediated GTPγS binding. The reaction was terminated by filtration through Whatman GF/C filter papers followed by washing with cold TME buffer. The filter paper sections were collected and radioactivity was measured by liquid scintillation counting.

Khurana L., Fu B.Q., Duddupudi A.L., Liao Y.H., Immadi S.S., Kendall D.A, & Lu D. (2017). Pyrimidinyl Biphenylureas: Identification of New Lead Compounds as Allosteric Modulators of the Cannabinoid Receptor CB1. Journal of medicinal chemistry, 60(3), 1089-1104.

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GTPγS Assay for CB1 Receptor Modulation

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To assess the impact of 3c and 3d on G-protein coupling of CB₁, GTPγS assays were performed essentially as described previously.^{20 (link)} Membranes of CB₁ expressing cells were prepared, and 8 μg of membrane preparation was incubated with 0.1 μM of CP55,940 plus or minus the allosteric modulator, allosteric modulator alone, or 1 μM SR141716A alone, and 0.1 nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), 10 μM GDP (Sigma, St. Louis, MO), and 0.1% (w/v) BSA. GTPγS binding assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl) was added to 200 μL. The membranes were incubated for 1 h at 30°C. To determine nonspecific binding, 10 μM unlabeled GTPγS (Sigma) was used. To determine basal activity, membrane preparations were treated with vehicle (dimethylsulfoxide or DMSO) alone. Termination of the reaction was achieved through filtration using Whatman GF/C filter papers and washing with cold TME buffer. Bound radioactivity was measured by liquid scintillation counting.

Immadi S.S., Dopart R., Wu Z., Fu B., Kendall D.A, & Lu D. (2018). Exploring 6-Azaindole and 7-Azaindole Rings for Developing Cannabinoid Receptor 1 Allosteric Modulators. Cannabis and Cannabinoid Research, 3(1), 252-258.

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GTPγS Binding Assay for Opioid Receptors

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Human MOR, DOR, or KOR recombinant CHO cell membranes were incubated for 2 h at 25 °C in 0.25 mL of 50 mM Tris–HCl buffer containing 5 mM MgCl₂, 1 mM EGTA, 100 mM NaCl, pH 7.4 with various concentrations of the tested compound, 30 μM guanosine 5-diphosphate (GDP) and 0.1 nM [³⁵S]GTPγS (PerkinElmer, Inc.). The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (Perkin Elmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). EC₅₀, E_max, and 95% CI values were calculated by Prism software (version 5.0). Nonspecific binding was measured in the presence of 10 μM unlabeled GTPγS (PerkinElmer, Inc.). DPDPE and ICI-174,864 (PerkinElmer, Inc.) were used as the standard DOR full agonist and full inverse agonist, respectively.

Iwamatsu C., Hayakawa D., Kono T., Honjo A., Ishizaki S., Hirayama S., Gouda H, & Fujii H. (2020). Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives. Molecules, 25(17), 3792.

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GTPγS Binding Assay for 2-AG Activity

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GTPγS binding assays were performed as described previously.^{62 (link)} Briefly, 6 μg of membranes was incubated for 60 min at 30 °C in GTPγS binding assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl) with the unlabeled 2-arachidonoylglycerol (2-AG) (at least nine different concentrations were used ranging between 100 pM and 1 μM), 0.1 nM [³⁵S]GTPγS (1250 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), 10 μM GDP (Sigma, St. Louis, MO), and 0.1% (w/v) BSA in the absence and presence of 10 μM test compounds. The effect of the compound on inhibiting the level of basal GTPγS binding was evaluated in the absence of agonist. Nonspecific binding was determined with 10 μM unlabeled GTPγS (Sigma, St. Louis, MO). After rapid filtration through Whatman GF/C filters the radioactivity trapped in the filters was determined by liquid scintillation counting.

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GLP-1R Activation Assay Using [35S]GTPγS Binding

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Preparation of GLP-1R HEK293 cell membranes and measurement of GLP-1R activation via [³⁵S]GTPγS binding to Gα_s using an antibody capture scintillation proximity assay were performed as described previously (20 (link)). Briefly, reactions contained 5 μg of membrane in 20 mm HEPES, pH 7.4, 50 mm NaCl, 5 mm MgCl₂, 40 μg/ml saponin, 0.1% bovine serum albumin, and 500 pm³⁵S-labeled GTPγS (PerkinElmer Life Sciences). Peptides and compounds were diluted and treated at a final concentration of 1% DMSO. Binding was induced for 30 min at room temperature before solubilization with 0.2% Nonidet P-40 detergent, rabbit anti-Gα_s polyclonal antibody, and 0.5 mg of anti-rabbit polyvinyl toluene beads (PerkinElmer Life Sciences). The detection mixtures were developed for 30 min, centrifuged at 80 × g for 10 min, and counted for 1 min/well using a MicroBeta TriLux instrument (PerkinElmer Life Sciences).

Bueno A.B., Showalter A.D., Wainscott D.B., Stutsman C., Marín A., Ficorilli J., Cabrera O., Willard F.S, & Sloop K.W. (2016). Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles. The Journal of Biological Chemistry, 291(20), 10700-10715.

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Measuring Opioid Receptor Activity

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Functional assays were conducted on hKOR-CHO cell membranes. Membranes were prepared in 50 mM Tris-HCl buffer pH 7.7. Cells were harvested by scraping the plates with a rubber policeman and were then centrifuged at 500× g for 10 min. The cell pellet was resuspended in Tris-HCl, homogenized with a Dounce homogenizer and centrifuged at 27,000× g for 15 min. The pellets were resuspended in Tris-HCl and homogenized through a 27G needle. The protein concentration of the homogenate was determined by Biuret reaction (RotiQuant Universal, Carl Roth) and stored at −70 °C until use.
To perform the GTPγS assay, membrane homogenates (10 μg/reaction tube) were diluted in HEPES buffer (20 mM) together with 10 μM GDP and 0.05 nM [³⁵S]GTPγS (Perkin Elmer, MA, USA). The buffer containing the cell membranes was then incubated with increasing concentration of test peptide (total volume of 1 mL) for 60 min at 25 °C. Non-specific binding was determined using 10 μM unlabeled GTPγS (Sigma-Aldrich, Vienna, Austria). Samples were filtered over glass fiber filters (GF/B Whatman) and the bound radioactivity was measured by liquid scintillation counting as counts per minute (CPM).

Lieb A., Thaler G., Fogli B., Trovato O., Posch M.A., Kaserer T, & Zangrandi L. (2021). Functional Characterization of Spinocerebellar Ataxia Associated Dynorphin A Mutant Peptides. Biomedicines, 9(12), 1882.

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