Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Abstract The glucagon‐like peptide‐1 receptor (GLP‐1R), a family B G‐protein coupled receptor (GPCR), regulates the insulin secretion following stimulation by ligands. The transmembrane domain (TM) mediates GLP‐1R homodimerization, which modulates its ligand binding and signaling. We investigated the possible involvement of the N‐terminal extracellular domain (NTD) in dimerization/oligomerization and dimer‐associated ligand binding by NanoLuc Binary Technology (NanoBiT). With improved NanoBiT detection using a decreasing substrate concentration, the negative cooperativity of ligand binding to the NTD was confirmed by accelerated dissociation and Scatchard analysis. The dimerization/oligomerization of the isolated NTD was observed by NanoBiT and validated by analytical ultracentrifugation, deriving the comparable dimerization affinity (~10 5 M −1 ). The NTD was also involved in the dimerization/oligomerization of the full‐length GLP‐1R with mutated TM4 at the cellular level. In an analysis of the parameters of the NTD binding, the K d for the probe GLP‐1 (7‐36, A8G) was similar (6‐8 μM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP‐1 and dulaglutide, exenatide showed two‐site binding with K i values of 1.4 pM and 8.7 nM. Our study indicates the involvement of NTD in the GLP‐1R dimerization/oligomerization and suggests that further investigations on the role in other family B GPCRs are needed.