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Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast.

Weston C, Poyner D, Patel V, Dowell S, Ladds G - Br. J. Pharmacol. (2014)

Bottom Line: This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity.We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway.These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK.

No MeSH data available.


Related in: MedlinePlus

The GPA1/Gαs responses reproduce cAMP data for GLP-1 receptor agonism. (A) Dose-response curves to the natural GLP-1 receptor agonist, GLP-1, were constructed in the yeast strain containing the GPA1/Gαs chimera. Activation of the reporter gene was calculated as a percentage of the maximum response observed. (B) cAMP accumulation was determined following 30 min stimulation of transiently transfected HEK293T cells and expressed as a percentage of the maximum observed. (C) S. cerevisiae containing the GPA1/Gαs chimera and (D) transiently transfected HEK293T cells were stimulated with GLP-1 in the presence of the indicated concentrations of exendin-3. All data are expressed as a percentage of the maximal response in the absence of inhibitor and are mean of 5–8 independent experiments ± SEM. (E) and (F) Double reciprocal plots for GLP-1 in the presence (Y axis) and absence (X axis) of 10 nM exendin-3.
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fig02: The GPA1/Gαs responses reproduce cAMP data for GLP-1 receptor agonism. (A) Dose-response curves to the natural GLP-1 receptor agonist, GLP-1, were constructed in the yeast strain containing the GPA1/Gαs chimera. Activation of the reporter gene was calculated as a percentage of the maximum response observed. (B) cAMP accumulation was determined following 30 min stimulation of transiently transfected HEK293T cells and expressed as a percentage of the maximum observed. (C) S. cerevisiae containing the GPA1/Gαs chimera and (D) transiently transfected HEK293T cells were stimulated with GLP-1 in the presence of the indicated concentrations of exendin-3. All data are expressed as a percentage of the maximal response in the absence of inhibitor and are mean of 5–8 independent experiments ± SEM. (E) and (F) Double reciprocal plots for GLP-1 in the presence (Y axis) and absence (X axis) of 10 nM exendin-3.

Mentions: GPCR specificity for individual yeast chimeras has previously been demonstrated to generally conform to that observed in mammalian cells, indeed the GLP-1 receptor has been reported to couple through Gαs, Gαi and Gαq in other systems (Baggio and Drucker, 2007). We next compared the pharmacology of the GLP-1 receptor with respect to GPA1/Gαs activation in yeast cells and accumulation of cAMP in transiently transfected HEK293T cells. Initially, concentration-response curves were constructed to the GLP-1 receptor agonist, GLP-1, in both yeast and mammalian cells expressing the receptor (Figure 2A and B). Sigmoidal dose-response curves were observed allowing the calculation of an EC50 value for GLP-1 (10 nM and 150 pM, yeast and mammalian assays respectively). Notably, the agonist was more potent for the accumulation of cAMP than in the yeast system; however, both responses were robust over five repeats with minimal error (Table 1).


Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast.

Weston C, Poyner D, Patel V, Dowell S, Ladds G - Br. J. Pharmacol. (2014)

The GPA1/Gαs responses reproduce cAMP data for GLP-1 receptor agonism. (A) Dose-response curves to the natural GLP-1 receptor agonist, GLP-1, were constructed in the yeast strain containing the GPA1/Gαs chimera. Activation of the reporter gene was calculated as a percentage of the maximum response observed. (B) cAMP accumulation was determined following 30 min stimulation of transiently transfected HEK293T cells and expressed as a percentage of the maximum observed. (C) S. cerevisiae containing the GPA1/Gαs chimera and (D) transiently transfected HEK293T cells were stimulated with GLP-1 in the presence of the indicated concentrations of exendin-3. All data are expressed as a percentage of the maximal response in the absence of inhibitor and are mean of 5–8 independent experiments ± SEM. (E) and (F) Double reciprocal plots for GLP-1 in the presence (Y axis) and absence (X axis) of 10 nM exendin-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128063&req=5

fig02: The GPA1/Gαs responses reproduce cAMP data for GLP-1 receptor agonism. (A) Dose-response curves to the natural GLP-1 receptor agonist, GLP-1, were constructed in the yeast strain containing the GPA1/Gαs chimera. Activation of the reporter gene was calculated as a percentage of the maximum response observed. (B) cAMP accumulation was determined following 30 min stimulation of transiently transfected HEK293T cells and expressed as a percentage of the maximum observed. (C) S. cerevisiae containing the GPA1/Gαs chimera and (D) transiently transfected HEK293T cells were stimulated with GLP-1 in the presence of the indicated concentrations of exendin-3. All data are expressed as a percentage of the maximal response in the absence of inhibitor and are mean of 5–8 independent experiments ± SEM. (E) and (F) Double reciprocal plots for GLP-1 in the presence (Y axis) and absence (X axis) of 10 nM exendin-3.
Mentions: GPCR specificity for individual yeast chimeras has previously been demonstrated to generally conform to that observed in mammalian cells, indeed the GLP-1 receptor has been reported to couple through Gαs, Gαi and Gαq in other systems (Baggio and Drucker, 2007). We next compared the pharmacology of the GLP-1 receptor with respect to GPA1/Gαs activation in yeast cells and accumulation of cAMP in transiently transfected HEK293T cells. Initially, concentration-response curves were constructed to the GLP-1 receptor agonist, GLP-1, in both yeast and mammalian cells expressing the receptor (Figure 2A and B). Sigmoidal dose-response curves were observed allowing the calculation of an EC50 value for GLP-1 (10 nM and 150 pM, yeast and mammalian assays respectively). Notably, the agonist was more potent for the accumulation of cAMP than in the yeast system; however, both responses were robust over five repeats with minimal error (Table 1).

Bottom Line: This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity.We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway.These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK.

No MeSH data available.


Related in: MedlinePlus