glp 1r plasmids  (Addgene inc)

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1R vs GIPR trafficking patterns. (A) Internalization AUC dose response curves from DERET assays in INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells stimulated with the indicated concentrations of GLP-1 or GIP, respectively. Results were fitted to a 3-parameter dose response curve to obtain Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 5. (B) Rates of GLP-1R vs GIPR internalization (k values) derived from (A) by one-phase association (with Y0 = 0) of baseline-deleted DERET data; n = 5. (C) GLP-1R vs GIPR internalization dose response curves measured by high-content microscopy assay (HCA) in the same cells as above. Results fitted and Emax and logEC50 comparisons included as above; n = 5. (D) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red, middle panels) localization following 30 minutes stimulation with 100 nM GLP-1-FITC or GIP-FITC (green, left panels), respectively, in the same cells as above. (E) Confocal microscopy analysis of isolated intact mouse islets stimulated with fluorescently labeled agonists as indicated: WT islets were imaged following stimulation with 100 nM GLP-1-TMR, while both WT and GIPR −/− (KO) islets were imaged following stimulation with 1 µM GIP-TMR. (F) Quantification of surface GLP-1R vs GIPR levels in WT mouse islets using fluorescent agonist uptake data from (E); data corrected for binding affinity differences between both agonists; n = 4. (G) GLP-1R vs GIPR recycling dose response curves measured by high-content microscopy assay (HCA) in cells from (C); n = 5. (H) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red, middle panels) colocalization with SNX27-GFP (green, left panels) following 3 hours stimulation with 100 nM GLP-1 or GIP, respectively, in the same cells as above. Nuclei (DAPI), blue. Data are mean ± SEM, compared by paired or unpaired t tests, or two-way ANOVA with Sidak's post hoc test; * P < .05, ** P < .01, *** P < .001; size bars: 10 µm.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Derivative Assay, Microscopy, Confocal Microscopy, Isolation, Labeling, Binding Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Endosomal vs plasma membrane localization of GLP-1R compared with GIPR in beta cells. (A) GLP-1R vs GIPR plasma membrane localization dose response curves from NanoBRET assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing KRAS-Venus and GLP-1R- or GIPR-NanoLuc, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; results were fitted to 3-parameter dose response curves to obtain Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 5. (B) As for (A) but for GLP-1R vs GIPR endosomal localization dose response curves from NanoBRET assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Rab5-Venus and GLP-1R- or GIPR-NanoLuc, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; n = 5. Data are mean ± SEM, compared by paired t tests or two-way ANOVA with Sidak's test; * P < .05.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: GLP-1R vs GIPR clustering propensities in beta cells. (A) Representative images from RICS analysis of GLP-1R vs GIPR clustering, showing SNAP-Surface 488-labeled GLP-1Rs or GIPRs imaged in the basolateral plane of INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells after 5 minutes treatment in vehicle (Veh) and either 100 nM GLP-1 or GIP. Diffusion coefficients for individual ROIs are indicated on each image, with corresponding intensity traces, as well as 2D and fitted 3D autocorrelation maps for each ROI, are also depicted. (B) Average RICS diffusion coefficients for each receptor and treatment for each cell analyzed from n = 4 experiments. (C) GLP-1R clustering kinetics measured by TR-FRET in INS-1 832/3 SNAP-GLP-1R cells treated with 100 nM agonist as indicated, with vehicle-corrected AUCs included; n = 4. (D) GIPR clustering kinetics measured by TR-FRET in INS-1 832/3 SNAP-GIPR cells treated with 100 nM agonist as indicated, with vehicle-corrected AUCs included; n = 4. Data are mean ± SEM, compared by one-way ANOVA with Sidak's test; **** P < .0001; ns: non-significant.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Labeling, Diffusion-based Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1r vs GIPR degradation propensities. (A) Western blot assessment of SNAP-GLP-1R or GIPR over tubulin levels in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for 6 hours in the presence of the protein synthesis inhibitor cycloheximide; n = 3. (B) Representative Western blot results from (A). Note that the top bands were used to quantify the SNAP-receptor levels, as they correspond to the glycosylated forms of the receptors, known to be biologically active and correctly inserted at the plasma membrane . (C) Percentage of GLP-1R vs GIPR, labeled with the cell-permeable SNAP-tag probe BG-OG, and corresponding representative images from INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for the indicated times in the presence of cycloheximide; n = 4. (D) Percentage of co-localization (Mander's coefficient) and representative images of SNAP-GLP-1R vs -GIPR (labeled with SNAP-Surface 649) with Lysotracker Green in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells stimulated with 100 nM GLP-1 or GIP for 1 hour; n = 5. Data are mean ± SEM, compared by ratio-paired or unpaired t test or two-way ANOVA with Sidak's post hoc test; *** P < .001, **** P < .0001.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Western Blot, Labeling

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1R vs GIPR G protein subtype and β-arrestin 2 recruitment characteristics. (A) Kinetics of Gα s , Gα q , Gα i , and β-arrestin 2 recruitment to the GLP-1R assessed by NanoBiT complementation assay in INS-1 832/3 GLP-1R KO cells transiently expressing GLP-1R-SmBiT and the corresponding mini-G protein subtype or β-arrestin 2. Responses to 100 nM GLP-1 normalized to vehicle and corresponding AUCs are shown; n = 5. (B) Kinetics of Gα s , Gα q , Gα i , and β-arrestin 2 recruitment to the GIPR assessed by NanoBiT complementation assay in INS-1 832/3 GIPR KO cells transiently expressing GIPR-SmBiT and the corresponding mini-G protein subtype or β-arrestin 2. Responses to 100 nM GIP normalized to vehicle and corresponding AUCs are shown; n = 5. (C) NanoBRET assessment of GLP-1R vs GIPR recruitment of Gα s , performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing mini-Gs-Venus and either GLP-1R- or GIPR-NanoLuc, after stimulation with 100 nM GLP-1 or GIP, respectively, with corresponding AUCs also shown; n = 4. Data are mean ± SEM, compared by paired t test; *** P < .05.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: GLP-1R vs GIPR endosomal vs plasma membrane activity in beta cells. (A) GLP-1R vs GIPR plasma membrane activity dose response curves from bystander NanoBiT signaling assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, LgBiT-CAAX and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; 30-minute AUC for each agonist concentration tested were fitted to 3-parameter dose response curves to obtain plasma membrane Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 4. (B) As for (A) but for GLP-1R vs GIPR endosomal activity in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, Endofin-LgBiT and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; n = 4. Data are mean ± SEM, compared by paired t tests; * P < .05, *** P < .001.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Activity Assay, Expressing, Concentration Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Functional analysis of GLP-1R vs GIPR signaling in beta cells. (A) cAMP dose response curves to GLP-1 vs GIP assessed in INS-1 832/3 cells by HTRF assay; results were fitted to 3-parameter dose response curves to obtain plasma membrane Emax and logEC50 for both receptors, depicted here combined as log(Emax/EC50) for each receptor; n = 5. (B) cAMP FRET responses to 100 nM GLP-1 or GIP from isolated and 4-hydroxytamoxifen-treated Pdx1-Cre ERT /CAMPER mouse islets; including agonist AUCs calculated for each receptor; n = 4. (C) INS-1 832/3 calcium responses (using the calcium indicator Cal520-AM) to 100 nM GLP-1 or GIP, including agonist AUCs calculated for 0-1 minute and 1-5 minutes responses for each receptor; n = 4. (D) Calcium responses to 100 nM GLP-1 vs GIP from purified WT mouse islets loaded with Cal520-AM, including agonist AUCs for each receptor; n = 4. (E) Insulin secretion responses to 100 nM GLP-1 vs GIP, expressed as fold increases to 11 mM glucose (G11) secretion levels from INS-1 832/3 WT ( n = 3), GLP-1R KO ( n = 5) and GIPR KO ( n = 4) cells. Data are mean ± SEM, compared by paired t tests; * P < .05; ns, nonsignificant.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Functional Assay, HTRF Assay, Isolation, Purification

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Summary of trafficking, coupling, and cellular outputs of GLP-1R vs GIPR in response to cognate agonists

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: Signaling and internalization properties of GLP-1R, GIPR, GCGR. A , mini-G s , -G i , -G q , and β-arrestin-2 recruitment responses in HEK293T cells transiently transfected with each SmBiT-tagged receptor and LgBiT-tagged effector and stimulated with 100 nM GLP-1, GIP, GCG or vehicle; n = 4 for GLP-1R and GCGR and n = 5 for GIPR. B , β-Arrestin-2 activation response in HEK293T cells transiently transfected with each receptor and NLuc-4myc-βarr2-CyOFP1 and stimulated with 100 nM GLP-1, GIP, GCG or vehicle, n = 6. C , internalization of each SNAP-tagged receptor in HEK293T cells stimulated with 100 nM GLP-1, GIP, GCG, or vehicle, measured by DERET, n = 4. D , time course showing internalization of each SNAP-tagged receptor in response to 100 nM GLP-1, GIP, or GCG, measured by reversible SNAP-tag labeling in transiently transfected HEK293T cells, n = 5. E , representative images of transiently transfected HEK293T cells expressing each SNAP-tagged receptor and labeled with BG-S-S-649 prior to treatment ±100 nM GLP-1, GIP, or GCG. Images were acquired before and after removal of residual surface BG-S-S-649 using Mesna; the scale bar represents 16 μm. F , representative high-resolution images from n = 2 experiments of HEK293T cells transiently expressing each SNAP-tagged receptor and labeled with SNAP-Surface-549 before treatment ±100 nM GLP-1, GIP, or GCG; the scale bar represents 8 μm. G , heatmap summary of signaling and internalization responses shown in this figure, normalized to the GLP-1R response and expressed as a log 2 fold change. β-Arrestin-2 recruitment (“rec,” Fig. 1 A ) and activation (“act,” Fig. 1 B ) responses are both shown. GCGR DERET response falls below the displayed range and is marked with “X.” See also Fig. S1 . Data are represented as mean ± SEM. DERET, diffusion-enhanced resonance energy transfer; GCG, glucagon; GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic polypeptide; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor.

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques: Transfection, Activation Assay, Labeling, Expressing, Diffusion-based Assay, Förster Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: GLP-1R, GIPR, and GCGR responses in the absence of β-arrestins. A , cAMP responses at 10 or 60 min to GLP-1, GIP, or GCG, in wildtype (wt) or dual β-arrestin knockout (βarr1/2 KO) HEK293 cells transiently transfected with the indicated SNAP-tagged receptor, normalized to forskolin (FSK, 10 μM) response, respectively, n = 5, 4-parameter fits shown. B , dose responses for PKA activation in wildtype or dual β-arrestin knockout HEK293 cells transiently cotransfected with each SNAP-tagged receptor and AKAR4-NES, calculated from FRET signal AUC and normalized to vehicle responses, three-parameter fits shown, n = 5. See also Fig. S2 . C , time course for ERK1/2 phosphorylation in wildtype or dual β-arrestin knockout HEK293 cells transiently transfected with each SNAP-tagged receptor and stimulated with 100 nM GLP-1, GIP or GCG, normalized to basal response. Time points compared by randomized block two-way ANOVA with Sidak’s test, n = 6. D , representative images showing internalization of each transiently transfected SNAP-tagged receptors in wildtype or dual β-arrestin knockout HEK293 cells, labeled with BG-S-S-649 prior to 100 nM agonist (or vehicle) treatment, with removal of residual surface receptor using Mesna where indicated; the scale bar represents 25 μm. Surface expression and ligand-induced internalization are quantified from n = 5 experiments, with comparisons by paired t tests. ∗ p < 0.05 by statistical test indicated in the text. Data are represented as mean ± SEM with individual replicates in some places. GCG, glucagon; GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic polypeptide; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor.

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques: Knock-Out, Transfection, Activation Assay, Blocking Assay, Labeling, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: Effect of β-arrestin deletion on agonist-induced cAMP responses in HEK293 cells

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: Effect of β-arrestin deletion on agonist-induced cytoplasmic PKA activation in HEK293 cells

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: Biased incretin analogs with N-terminal region substitutions. A , cAMP responses in PathHunter CHO-GLP-1R, CHO-GIPR, or CHO-GCGR cells (as appropriate) to analogs of GLP-1, GIP, and GCG, 30-min stimulation, n = 5, 4-parameter fits shown. B , as for ( A ) but β-arrestin-2 recruitment responses. C , the relative impact of each amino acid substitution on cAMP signaling is shown by subtracting Log τ/K A for the reference agonist (GLP-1, GIP, or GCG) from that of each analog on a per-assay basis. D , As for ( C ) but for β-arrestin-2 recruitment. E , biased agonism for N-terminally modified GLP-1, GIP, and GCG analogs at their cognate receptors, calculated as normalized log transduction ratios [ΔΔlog(τ/K A )] relative to GLP-1, GIP, or GCG, respectively. The numerical degree of bias is indicated for each ligand after anti-log transformation. F , representative images showing GLP-1R and GIPR internalization after 30-min stimulation with indicated agonist at 100 nM, with quantification below from n = 5 experiments and comparison by one-way randomized block ANOVA with Dunnett’s test versus GLP-1 or GIP (as appropriate); the scale bar represents 26 μm. G , SNAP-GLP-1R or SNAP-GIPR internalization in CHO-K1 cells treated with the indicated agonist (100 nM), measured by DERET, n = 4, AUC versus GLP-1 or GIP compared by randomized block one-way ANOVA with Dunnett’s test. ∗ p < 0.05 by statistical test indicated in the text. Data are represented as mean ± SEM (with individual replicates in some cases), except for bias plots where error bars indicate 95% confidence intervals. GCG, glucagon; GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic polypeptide; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor.

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques: Modification, Transduction, Transformation Assay, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Genetic and biased agonist-mediated reductions in β-arrestin recruitment prolong cAMP signaling at glucagon family receptors

doi: 10.1074/jbc.RA120.016334

Figure Lengend Snippet: Responses in INS-1 832/3 cells, Huh7 cells, and primary hepatocytes. A expression of Glp1r , Gipr , and Gcgr in INS-1 832/3 cells, determined by qPCR, normalized to expression of endogenous control gene 18S by 2 −ΔCt , n = 2. B , insulin secretory responses in INS-1 832/3 cells treated for 16 h with GLP-1 analogs at 11 mM glucose (“G11”), n = 5, three-parameter fits shown. C , As for ( B ) but with GIP analogs. D , correlation of GLP-1 and GIP analog maximum insulin secretion and maximum β-arrestin-2 recruitment ( Fig. 3 ) by linear regression. E , insulin secretory responses in wildtype and GLP-1R KO INS-1 832/3 cells treated for 16 h with GCG analogs at 11 mM glucose, n = 5, three-parameter fits shown. F , cAMP responses to GCG analogs in Huh7-GCGR cells treated for 16 h, relative to response to forskolin (10 min, 10 μM), n = 4, four-parameter fits shown. G , effect of prolonged (16-h) exposure to indicated agonist (10 nM) on upregulation of G6pc in Huh7-GCGR cells, n = 4. H , effect of prolonged (16-h) exposure to indicated agonist (100 nM) on glucose production by primary mouse hepatocytes, n = 4, expressed as fold change to vehicle stimulation. For ( G ) and ( H ), no treatment response was significantly different from that of GCG, by one-way randomized block ANOVA with Dunnett’s test. Data are represented as mean ± SEM and individual replicates in some cases. GCG, glucagon; GCGR, glucagon receptor; GIP, glucose-dependent insulinotropic polypeptide; GIPR, glucose-dependent insulinotropic polypeptide receptor; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor.

Article Snippet: Construction of the GLP-1R-SmBiT plasmid was described previously , and the same strategy was used to develop GIPR-SmBiT and GCGR-SmBiT, with cloning in frame at the C terminus of the receptor by substitution of the Tango sequence on a FLAG-tagged GPCR-Tango expression vector , a gift from Dr Bryan Roth, University of North Carolina (Addgene # 66295).

Techniques: Expressing, Blocking Assay