In Vitro Characterization of Agonist and Antagonist Peptide Binding Interaction Kinetics to GLP-1R in HEK293T Cells...

The glucagon peptide 1 receptor (GLP-1R), a class-B-type G protein-coupled receptor (GPCR), is a key therapeutic target for many metabolic disorders, including obesity and type 2 diabetes, due to its central role in glucose homeostasis and insulin secretion. Despite its pharmacological importance, studying the binding kinetics of its multidomain engagement with peptide ligands remains a challenge using purified receptor systems. The isolated forms fail to capture the dynamic behavior of membrane-bound GPCRs in a physiologically relevant context. A deeper understanding of the interaction kinetics of agonist and antagonist binding to GLP-1R domains is essential for rational drug design, as the activation of the receptor depends on distinct binding modes that modulate downstream signaling efficacy. Here we employ surface plasmon resonance microscopy (SPRM) on HEK293T cells overexpressing GLP-1R to visualize and quantify the label-free kinetic interactions of ligands on whole single cells in real time. Using three different agonists (GLP-1, liraglutide, exendin-4) and one antagonist (exendin-9), we demonstrate that the agonists exhibit a two-mode/bivalent binding behavior with C-terminal engagement of the extracellular domain (ECD) and N-terminal engagement of the transmembrane domain (TMD). In contrast, the antagonist exendin-9 binds with a single mode, exclusively to the ECD. Importantly, SPRM resolves not only the presence of dual-domain engagement but also the stability and heterogeneity of these interactions, enabling discrimination between full and partial agonism. Notably, liraglutide displays the highest interaction affinity and the greatest amount of activation through TMD binding, which agrees with its known structural optimization and superior therapeutic performance. This study highlights SPRM as a powerful, label-free platform for probing the on-cell binding kinetics of GPCR interactions with peptides, providing quantitative insights into the activation efficiency of agonists and selectivity of antagonists in ways conventional receptor assays cannot.