Spatiotemporal GLP-1 and GIP receptor signaling and trafficking/recycling dynamics induced by selected receptor mono- and dual-agonists

Objective We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. Methods Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. Results Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. Conclusions Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.
Highlights • GLP-1/GIP dual-agonists, MAR709 and tirzepatide, are partial effectors at multiple GLP-1R pathways, yet retain full cAMP agonism. • MAR709 elicits comparable GLP-1R incorporation into Rab11+ recycling endosomes relative to the native peptides and acyl-GLP-1. • At the GIPR, both dual-agonists exhibit full-agonism properties with limited receptor internalization/trafficking properties.