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 (~105 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 Kd for the probe GLP-1 (7-36, A8G) was similar (6-8μM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP-1 and dulaglutide, exenatide showed two-site binding with Ki values of 1.4pM and 8.7nM. 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.