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 Gα_s 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.