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

Abstract

Background and PurposeThe glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities.Experimental ApproachWe describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. 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.Key ResultsThe GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. 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. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor.Conclusions and ImplicationsThese 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.

A Hydrogen-Bonded Polar Network in the Core of the Glucagon-Like Peptide-1 Receptor Is a Fulcrum for Biased Agonism: Lessons from Class B Crystal Structures

“…Whereas GLP-1 and exendin-4 have similar biases for these pathways at the wild-type receptor, there is increasing evidence from mutational studies that these peptides have distinct modes of receptor activation (Koole et al, 2012a andb, Wootten et al, 2013b), and this is also supported by differences in peptide-mediated activation of G protein chimeras in yeast (Weston et al, 2014). The current study provides additional evidence for how these peptides use the central polar network to drive activation of the receptor.…”

“…Nonetheless, class B GPCRs have their own unique set of conserved, intramembranous, polar residues that are likely comparable to those in class A. Prototypical of this receptor class is the GLP-1 receptor that displays pleiotropic coupling and both peptide-and nonpeptidic-biased agonism (Jorgensen et al, 2007;Coopman et al, 2010;Koole et al, 2010;Cheong et al, 2012;Willard et al, 2012;Wootten et al, 2013a;Weston et al, 2014). Recently, the role of the conserved intramembranous polar residues in this receptor was probed by alanine scanning mutagenesis, which revealed clusters of amino acids important for a range of functions, including protein expression and the control of activation transition for both general signal pathway bias and ligand-directed biased signaling (Wootten et al, 2013b).…”

Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity

“…We have previously used the methods developed by Figuero et al 37 to quantify ligand bias for receptors expressed in yeast. 19,20 Here we report the adaptation of the equimolar method of comparison 37 to quantify a ligands selectivity for a given receptor (see Experimental Section for more details). Since NECA is a full agonist for all three ARs expressed in yeast, it can be used as a reference ligand.…”

Optical Control of Insulin Secretion Using an Incretin Switch

“…Intriguingly, signal bias could be introduced depending on isomerization status, most likely owing to the pronounced effects of azobenzene orientation on peptide structure. This phenomenon is well‐reported for the GLP‐1R15, 18 and forms the basis of intense research efforts, since drug side effects may stem from presently unknown signaling interactions 19. The GLP‐1R is coupled to multiple pathways (e.g., cAMP, PKA, Epac2, ERK, and β‐arrestin), however, orthosteric ligands can provoke different receptor conformations to engage distinct signals 15.…”