Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 1. Activation of ERK-1/2 by wild-type M3-muscarinic re- ceptors and the deletion mutant DLys370–Ser425. Stably trans- fected CHO cells expressing either the wild-type human M3-muscarinic receptor or the deletion mutant DLys370–Ser425 were stimulated for 5 min in the presence of varying concentrations of carbachol (CCH). The reaction was terminated by addition of lysis buffer, and ERK-1/2 activ- ity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the DLys370–Ser425 receptor mutant. The data presented represent the mean 6 S.E. for three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Activation Assay, Mutagenesis, Stable Transfection, Expressing, Lysis, Concentration Assay, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 2. Serum-mediated ERK-1/2 responses. Stably transfected CHO cells expressing either the wild-type human M3-muscarinic recep- tor or the deletion mutant DLys370–Ser425 were stimulated for 20 min in the presence of varying concentrations of fetal calf serum. The reaction was terminated by addition of lysis buffer, and ERK-1/2 activity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the DLys370–Ser425 receptor mutant. The data represent the mean 6 S.E. of at least three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Stable Transfection, Transfection, Expressing, Mutagenesis, Lysis, Activity Assay, Concentration Assay, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 5. Effect of the CK1a dominant negative mutant (F- CK1aK46R) and the 3i-loop peptide on the M3-muscarinic ERK- 1/2 response. CHO-m3 cells stably expressing recombinant M3-mus- carinic receptors (A) or native CHO-K1 cells (B), were transiently transfected with the CK1a dominant negative mutant, F-CK1aK46R (K46R) or the 3i-loop peptide (3i-loop) corresponding to Ser345–Leu463 of the third intracellular loop of the M3-muscarinic receptor or were sham transfected (Control). 48 h after transfection, cells were stimulated with 1 mM carbachol (CCH, A) or 1 mM phorbol 12,13-dibutyrate (PDBu, B), for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean 6 S.E. of three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Dominant Negative Mutation, Stable Transfection, Expressing, Recombinant, Transfection, Control, Lysis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 6. ERK-1/2 concentration-response curves in CHO-m3 cells transiently transfected with the CK1a dominant negative mutant (F-CK1aK46R) and the 3i-loop peptide. CHO-m3 cells sta- bly expressing recombinant M3-muscarinic receptors were transiently transfected with the CK1a dominant negative mutant, F-CK1aK46R (K46R) (A), or the 3i-loop peptide (3i-loop peptide, B) corresponding to Ser345–Leu463 of the third intracellular loop of the M3-muscarinic re- ceptor. 48 h after transfection cells were stimulated with varying con- centrations of carbachol (CCH) for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean 6 S.E. of three experiments.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Concentration Assay, Transfection, Dominant Negative Mutation, Expressing, Recombinant, Lysis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway

doi: 10.1074/jbc.m008827200

Figure Lengend Snippet: FIG. 7. Scheme of the mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors. Our data have identified two mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors expressed in CHO cells. Mecha- nism 1 is PKC-dependent and is essential in the activation of ERK-1/2. Inhibition of Mechanism 1 (e.g. inhibition of PKC with Ro-318220) prevents activation of ERK-1/2 despite the fact that Mechanism 2 is still intact. Mechanism 2, therefore, will not elicit an ERK-1/2 response alone. However, Mechanism 2 does operate in concert with Mechanism 1 to give a full ERK-1/2 response. Hence a receptor that is only able to activate Mechanism 1 (i.e. the DLys370–Ser425 receptor mutant or the wild-type M3-muscarinic receptor expressed together with the 3i-loop peptide or F-CK1aK46R) will give a less than maximal ERK-1/2 re- sponse. CK1a, casein kinase 1a; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

Article Snippet: Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 mg of anti-Erk-1/2 antiserum (Santa Cruz).

Techniques: Activation Assay, Inhibition, Mutagenesis

Journal: Metabolites

Article Title: The Cholesterol Metabolite Cholest-5-en-3-One Alleviates Hyperglycemia and Hyperinsulinemia in Obese ( db / db ) Mice

doi: 10.3390/metabo12010026

Figure Lengend Snippet: Luciferase activities in NFκB-Luc/CHO-K1 cells. Cells were treated with or without murine TNFα (10 ng/mL in DMSO), cholesterol (100 µM in DMSO), 5-cholestenone (100 µM choles-5-en-3-one in DMSO), or 1 µM IKK2 inhibitor IV. After 7 h of incubation, NFκB luciferase was detected using the ONE-Step TM Luciferase assay system. Values are expressed as the mean ± standard error ( n = 3 wells). * Significant difference at p < 0.05 compared to DMSO (TNFα-) treatment. † Significant difference at p < 0.05 compared to DMSO (TNFα+) treatment.

Article Snippet: In the NFκB-Luc/CHO-K1 cell line (BPS Bioscience, San Diego, CA, USA), fLUC expression is controlled by the NFκB response element located upstream of the TATA promoter and is suitable for monitoring the activity of the NFκB transcription factor through luminescence readout.

Techniques: Luciferase, Incubation

Journal:

Article Title: Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G)

doi: 10.1194/jlr.M800625-JLR200

Figure Lengend Snippet: Phenolic acids stimulate 35S-GTPγS binding to membranes from cells transfected with GPR109A. Ligand-induced 35S-GTPγS binding was studied using membranes prepared from CHO-K1 cells expressing human GPR109A (A), GPR109B (B), or GPR81 (C). Binding of 35S-GTPγS was determined in the presence of increasing concentrations of nicotinic acid (closed circles), acifran (triangles), trans-cinnamic acid (closed diamonds), p-coumaric acid (open circles), o-coumaric acid (squares), benzoic acid (open diamonds), or salicylic acid (crosses). Data are shown as the means ± SD of triplicates of a representative experiment. Three to four independent experiments were performed with similar results.

Article Snippet: Membranes prepared from Chinese Hamster Ovary (CHO)-K1 cells stably expressing GPR109A, GPR109B, GPR81, PUMA-G, or vector control (7 μg/assay) were diluted in assay buffer (100 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4) in Wallac Scintistrip plates (PerkinElmer, Shelton, CT) and preincubated with test compounds diluted in assay buffer containing 40 μM GDP (final [GDP] was 10 μM) for ∼10 min before addition of 35 S-GTPγS to 0.3 nM.

Techniques: Binding Assay, Transfection, Expressing

Journal:

Article Title: Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G)

doi: 10.1194/jlr.M800625-JLR200

Figure Lengend Snippet: Phenolic acids compete with 3H-nicotinic acid binding to membranes from cells transfected with GPR109A. Binding of 50 nM 3H-nicotinic acid to membranes prepared from CHO-K1 cells expressing human GPR109A was studied in the presence of increasing concentrations of A: nicotinic acid (closed circles) or the cinnamic-acid-related compounds, including trans-cinnamic acid (closed squares), p-coumaric acid (open circles), o-coumaric acid (open squares), and caffeic acid (crosses), or B: nicotinic acid (closed circles) or the benzoic-acid-related compounds, including benzoic acid (closed triangles), p-hydroxybenzoic acid (open triangles), salicylic acid (open diamonds), and protocatechuic acid (closed diamonds). Data are shown as the means ± SD of triplicates of a representative experiment.

Article Snippet: Membranes prepared from Chinese Hamster Ovary (CHO)-K1 cells stably expressing GPR109A, GPR109B, GPR81, PUMA-G, or vector control (7 μg/assay) were diluted in assay buffer (100 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4) in Wallac Scintistrip plates (PerkinElmer, Shelton, CT) and preincubated with test compounds diluted in assay buffer containing 40 μM GDP (final [GDP] was 10 μM) for ∼10 min before addition of 35 S-GTPγS to 0.3 nM.

Techniques: Binding Assay, Transfection, Expressing