Fig 1.
Small molecule agonists induce cAMP production but not intracellular Ca2+ accumulation or ERK phosphorylation in hGLP-1R expressing cells.
HEK293 cells cotransfected with the hGLP-1R plasmid and the luciferase reporter plasmid for cAMP (pGL4.29-Luc-CRE), intracellular Ca2+ (pGL4.30-Luc-NFAT) or ERK phosphorylation (pGL4.33-Luc-SRE) were stimulated with GLP-1, compound 2 or compound B as indicated to assess cAMP production (A), intracellular Ca2+ accumulation (Ca2+i) (B) and ERK phosphorylation (ERK-p) (C). (D) The EC50 of GLP-1, compounds 2 and B for cAMP production, (Ca2+i) and ERK-p (n.d., not determined). Data normalised to percentage stimulation of GLP-1 and are shown as mean ± SEM, n = 3.
Fig 2.
Concentration dependent stimulation of hGLP-1R internalisation by GLP-1, compound 2 and compound B.
HEK293 cells expressing the hGLP-1R were stimulated with GLP-1, compound 2 or compound B at the indicated concentration for 60min and hGLP-1R internalisation was assessed by ELISA using the anti-GLP-1R antibody (A) and the VSVG-antibody (B) and by immunofluorescence (D). (C) The EC50 of GLP-1, compounds 2 and B for hGLP-1R internalisation (n.d., not determined). In immunofluorescence, GFP-tagged hGLP-1R (green) and the anti-GLP-1R antibody staining (red) overlay is shown in yellow and nuclear staining with DAPI in blue. Data are mean ± SEM, n = 3.
Fig 3.
Time dependent stimulation of hGLP-1R internalisation by GLP-1, compound 2 and compound B.
HEK293 cells expressing the hGLP-1R were stimulated with 100nM GLP-1, 10μM compound 2 or 10μM compound B for the indicated time and assessed hGLP-1R internalisation by ELISA using the anti-GLP-1R antibody (A) and live cell fluorescence imaging (B). Data are mean + SEM, n = 3 (A) or representative of three independent experiments (B).
Fig 4.
Antagonists Ex(9–39) and JANT-4 inhibit cAMP production induced by GLP-1 but not compound 2 or compound B.
HEK293 cells cotransfected with the hGLP-1R and the luciferase reporter plasmid for cAMP (pGL4.29-Luc-CRE) were stimulated with GLP-1 (A), compound 2 (B) or compound B (C), as indicated, in the presence of 100nM Ex(9–39) (left panel) or JANT-4 (right panel) to assess cAMP production. (D) The EC50 of GLP-1, compounds 2 and B in the presence or absence of Ex(9–39) or JANT-4 for cAMP production. Data are mean ± SEM, n = 3, *** p <0.001.
Fig 5.
Concentration dependent stimulation of hGLP-1R internalisation by GLP-1 in the presence of antagonists Ex(9–39) and JANT-4.
HEK293 cells expressing the hGLP-1R were stimulated with GLP-1 at the indicated concentration for 60min in the presence of 100nM Ex(9–39) (left panel) or JANT-4 (right panel) and the internalisation of hGLP-1R was assessed by ELISA (A) and immunofluorescence (C) using the anti-GLP-1R antibody. (B) The EC50 of GLP-1 in the presence or absence of Ex(9–39) or JANT-4 for hGLP-1R internalisation. In immunofluorescence, GFP-tag of hGLP-1R (green) and the anti-GLP-1R antibody staining (red) overlay is shown in yellow and nuclear staining with DAPI in blue. Data are mean + SEM, n = 3, ** p<0.01; *** p <0.001.
Fig 6.
Effect of V36A and K334A mutations of the hGLP1R on the receptor cell surface expression and activity.
HEK293 cells were transfected with the hGLP-1R WT (wild-type) or the V36A or K334A mutants for 48h. (A) Total protein expression was assessed by immunoblotting using the anti-GFP and anti-VSVG antibodies. Cell surface expression (B) and agonist induced internalisation (C) were assessed by ELISA using the anti-GLP-1R antibody. (D) Immunofluorescence showing agonist induced internalisation of the WT or mutant hGLP-1R, GFP-tag of the constructs (green) and the anti-GLP-1R antibody staining (red) overlay is shown in yellow and nuclear staining with DAPI in blue. Data are mean + SEM, n = 3, n.s. p<0.05; *** p <0.001.
Fig 7.
Effect of V36A and K334A mutations of the hGLP-1R on the receptor induced cAMP production.
HEK293 cells cotransfected with the V36A (left panel) or K334A (right panel) hGLP-1R plasmid and the luciferase reporter plasmid for cAMP (pGL4.29-Luc-CRE) were stimulated with GLP-1 (A), compound 2 (B) or compound B (C) as indicated to assess cAMP production. (D) The EC50 of GLP-1, compounds 2 and B for cAMP production in cells expressing hGLP-1R WT or V36A or K334A (n.d., not determined). Data are mean ± SEM, n = 3, * p<0.05; ** p<0.01; *** p <0.001.
Fig 8.
Compounds 2 and B reduce the hGLP-1R internalisation by GLP-1.
HEK293 cells expressing the hGLP-1R were pre-incubated with either 10μM compound 2 or B for 60min and then stimulated with GLP-1 at the indicated concentration for a further 60min in the presence of 10μM compound 2 (left panel) or compound B (right panel) and hGLP-1R internalisation was assessed by ELISA (A) and immunofluorescence (B) using the anti-GLP-1R antibody. In immunofluorescence, GFP-tag of hGLP-1R (green) and the anti-GLP-1R antibody staining (red) overlay is shown in yellow and nuclear staining with DAPI in blue. Data are mean ± SEM, n = 3, *** p <0.001.
Fig 9.
Compounds 2 and B reduce GLP-1 stimulated intracellular Ca2+ accumulation and ERK phosphorylation in hGLP-1R expressing cells.
HEK293 cells cotransfected with the hGLP-1R plasmid and the luciferase reporter plasmid for cAMP (pGL4.29-Luc-CRE), intracellular Ca2+ (pGL4.30-Luc-NFAT) or ERK phosphorylation (pGL4.33-Luc-SRE) were pre-incubated with either 10μM compound 2 or B for 60min. Cells were then stimulated with GLP-1 at the indicated concentrations in the presence of 10μM compound 2 (left panel) or compound B (right panel) to assess cAMP production (A), intracellular Ca2+ accumulation (B) and ERK phosphorylation (C). Data normalised to percentage stimulation of GLP-1 and are shown as mean ± SEM, n = 3 (n.s. p>0.05; *** p <0.001).
Fig 10.
Schematic representation of the downstream signalling pathways activated by GLP-1 and small molecule agonist compounds 2 and B deduced from this study (Ca2+i, intracellular Ca2+ accumulation).