Modelling G protein-biased agonism using GLP-1 receptor C-terminal mutations
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Background and aim: The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target for type 2 diabetes and obesity. Agonists showing bias in favour of G protein signalling over β-arrestin recruitment and GLP-1R internalisation, e.g. tirzepatide and orforglipron, have favourable clinical efficacy profiles. However, understanding of the effects of biased agonism has been hampered by differences in ligand properties such as affinity, efficacy, stability and pharmacokinetics. Here we used GLP-1R C-tail mutations that inhibit phosphorylation to mimic G protein-biased GLP-1R agonism without the need for ligand modifications.
Methods: Serine doublet phosphorylation sites in the human and mouse GLP-1R C-tails were mutated to alanine. Wild-type and mutant GLP-1Rs were examined for β-arrestin recruitment, internalisation, Gαs activation, and signalling readouts in HEK293 cells and pancreatic β-cell models. Native GLP-1 plus oppositely biased ligands exendin-phe1 (ExF1; G protein-biased) and exendin-asp3 (ExD3; β-arrestin-biased) were used to compare ligand- and receptor-mediated biased agonism.
Results: Loss of three C-terminal phosphorylation sites reduced GLP-1- and ExD3-mediated GLP-1R internalisation and β-arrestin recruitment to that seen with ExF1. The phosphodeficient GLP-1R showed preferential plasma membrane Gαs activation over longer stimulations, with associated increases in whole cell cAMP generation and kinomic signalling. The distal GLP-1R phosphorylation site played a larger role in β-arrestin recruitment, and the proximal sites were more important for GLP-1R internalisation and regulating cAMP production.
Conclusion: Genetic changes that reduce in β-arrestin recruitment and slow GLP-1R internalisation can enhance GLP-1R signalling, providing conceptual support for the use of G protein bias to improve GLP-1R agonist efficacy.
Methods: Serine doublet phosphorylation sites in the human and mouse GLP-1R C-tails were mutated to alanine. Wild-type and mutant GLP-1Rs were examined for β-arrestin recruitment, internalisation, Gαs activation, and signalling readouts in HEK293 cells and pancreatic β-cell models. Native GLP-1 plus oppositely biased ligands exendin-phe1 (ExF1; G protein-biased) and exendin-asp3 (ExD3; β-arrestin-biased) were used to compare ligand- and receptor-mediated biased agonism.
Results: Loss of three C-terminal phosphorylation sites reduced GLP-1- and ExD3-mediated GLP-1R internalisation and β-arrestin recruitment to that seen with ExF1. The phosphodeficient GLP-1R showed preferential plasma membrane Gαs activation over longer stimulations, with associated increases in whole cell cAMP generation and kinomic signalling. The distal GLP-1R phosphorylation site played a larger role in β-arrestin recruitment, and the proximal sites were more important for GLP-1R internalisation and regulating cAMP production.
Conclusion: Genetic changes that reduce in β-arrestin recruitment and slow GLP-1R internalisation can enhance GLP-1R signalling, providing conceptual support for the use of G protein bias to improve GLP-1R agonist efficacy.
Date Issued
2026-03-01
Date Acceptance
2026-01-12
Citation
Molecular Metabolism, 2026, 105 (1)
ISSN
2212-8778
Publisher
Elsevier
Journal / Book Title
Molecular Metabolism
Volume
105
Issue
1
Copyright Statement
© 2026 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
License URL
Identifier
10.1016/j.molmet.2026.102321
Publication Status
Published
Article Number
102321
Date Publish Online
2026-01-20