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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

Comparison of GLP-1 receptor peptide ligands and relative bias factors. Sequences of the various peptide ligands for the GLP-1 receptor aligned to the potent, natural agonist. Amino acids differing from those in GLP-1 are highlighted in grey. The relative (to GLP-1) bias factor was quantified for each ligand as the change in log(τ/KA) ratio where a negative value indicates preference for the inhibitory, Gαi chimera. Statistical significance was determined using one-way anova with Bonferroni's post-test with each data set compared with GLP-1 (**P < 0.01, ***P < 0.001). Data are mean of 5–8 independent experiments ± SEM.
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fig08: Comparison of GLP-1 receptor peptide ligands and relative bias factors. Sequences of the various peptide ligands for the GLP-1 receptor aligned to the potent, natural agonist. Amino acids differing from those in GLP-1 are highlighted in grey. The relative (to GLP-1) bias factor was quantified for each ligand as the change in log(τ/KA) ratio where a negative value indicates preference for the inhibitory, Gαi chimera. Statistical significance was determined using one-way anova with Bonferroni's post-test with each data set compared with GLP-1 (**P < 0.01, ***P < 0.001). Data are mean of 5–8 independent experiments ± SEM.

Mentions: While several studies have investigated the effect of different ligands on the biasing of GLP-1 receptor signal transduction via calcium (Gαq-mediated) or ERK pathways (Koole et al., 2010), little has been reported for the inhibition of cAMP production. Since adverse side effects from the use of the GLP-1 mimetics could be attributed to previously unidentified off-target signalling events, we next used the yeast system to investigate ligand-induced signalling bias of other GLP-1 receptor ligands (Figure 8). The glucagon agonists, oxyntomodulin and glucagon successfully activated both the stimulatory and inhibitory G protein pathways via the GLP-1 receptor. However, both ligands displayed significant (P < 0.001, n = 5) bias towards Gαi. Similarly, the GLP-1 mimetic exenatide preferentially activated the inhibitory subunit. In contrast, only a small change in bias profile from the natural agonist was observed when liraglutide was used. Despite differences in peptide sequence primarily occurring at the C-terminus, with the exception of liraglutide, all ligands demonstrated a significant (P < 0.05, n = 5) degree of Gαi bias relative to the GLP-1. These data offer an intriguing insight into the mechanism by which peptide ligands activate family B receptors. Previous models suggest that the C-terminus is solely responsible for receptor binding to facilitate interaction of the N-terminus, which promotes activation (Hoare, 2005). Our results suggest that the C-termini of the peptides are primarily responsible for affecting the G protein preference of the activated receptor. However, given that the only peptide ligand tested that did not show a Gαi bias (liraglutide) also had the most highly conserved N-terminus, we cannot exclude the possibility that the N-terminus also plays a role in receptor signalling.


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)

Comparison of GLP-1 receptor peptide ligands and relative bias factors. Sequences of the various peptide ligands for the GLP-1 receptor aligned to the potent, natural agonist. Amino acids differing from those in GLP-1 are highlighted in grey. The relative (to GLP-1) bias factor was quantified for each ligand as the change in log(τ/KA) ratio where a negative value indicates preference for the inhibitory, Gαi chimera. Statistical significance was determined using one-way anova with Bonferroni's post-test with each data set compared with GLP-1 (**P < 0.01, ***P < 0.001). Data are mean of 5–8 independent experiments ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig08: Comparison of GLP-1 receptor peptide ligands and relative bias factors. Sequences of the various peptide ligands for the GLP-1 receptor aligned to the potent, natural agonist. Amino acids differing from those in GLP-1 are highlighted in grey. The relative (to GLP-1) bias factor was quantified for each ligand as the change in log(τ/KA) ratio where a negative value indicates preference for the inhibitory, Gαi chimera. Statistical significance was determined using one-way anova with Bonferroni's post-test with each data set compared with GLP-1 (**P < 0.01, ***P < 0.001). Data are mean of 5–8 independent experiments ± SEM.
Mentions: While several studies have investigated the effect of different ligands on the biasing of GLP-1 receptor signal transduction via calcium (Gαq-mediated) or ERK pathways (Koole et al., 2010), little has been reported for the inhibition of cAMP production. Since adverse side effects from the use of the GLP-1 mimetics could be attributed to previously unidentified off-target signalling events, we next used the yeast system to investigate ligand-induced signalling bias of other GLP-1 receptor ligands (Figure 8). The glucagon agonists, oxyntomodulin and glucagon successfully activated both the stimulatory and inhibitory G protein pathways via the GLP-1 receptor. However, both ligands displayed significant (P < 0.001, n = 5) bias towards Gαi. Similarly, the GLP-1 mimetic exenatide preferentially activated the inhibitory subunit. In contrast, only a small change in bias profile from the natural agonist was observed when liraglutide was used. Despite differences in peptide sequence primarily occurring at the C-terminus, with the exception of liraglutide, all ligands demonstrated a significant (P < 0.05, n = 5) degree of Gαi bias relative to the GLP-1. These data offer an intriguing insight into the mechanism by which peptide ligands activate family B receptors. Previous models suggest that the C-terminus is solely responsible for receptor binding to facilitate interaction of the N-terminus, which promotes activation (Hoare, 2005). Our results suggest that the C-termini of the peptides are primarily responsible for affecting the G protein preference of the activated receptor. However, given that the only peptide ligand tested that did not show a Gαi bias (liraglutide) also had the most highly conserved N-terminus, we cannot exclude the possibility that the N-terminus also plays a role in receptor signalling.

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