2.5. cAMP Accumulation Assay

cAMP accumulation was measured using the LANCE Ultra cAMP kit (PerkinElmer, Boston, MA, USA) according to the manufacturer’s instructions. CHO-K1 cells stably expressing wild-type (WT) or mutant GLP-1R were digested by 0.2% (w/v) EDTA and washed once with PBS. Cells were then resuspended with assay buffer (DMEM, 0.1% BSA, 5 mM HEPES) and seeded onto 384-well plates (6 × 10⁵/mL, 5 μL/well). Transfected cells were incubated for 40 min with 20 μM allosteric modulators and different concentrations of GLP-1. After the addition of 5 μL of Eu-cAMP tracer and ULight-anti-cAMP, the reactions were stopped. The plates were left for 1 h at room temperature to measure time-resolved FRET signals at excitation wavelengths of 620 nm and 665 nm by EnVision (PerkinElmer, Waltham, MA, USA). The cAMP response is depicted relative to the maximal response of GLP-1 at the WT receptor (100%).