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 (6‐8 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 Ki 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.

The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

“…It should be noted that, once occupancy differences are taken into account, exendin-4-like ligands show a comparative signalling deficit, as greater affinity did not fully translate into commensurate increases in signalling potency. A recent report describes the detection of two separate binding sites for exendin-4 on the GLP-1R extracellular domain (ECD), potentially in keeping with previous photoaffinity cross-linking data (34) and apparently responsible for impaired receptor oligomerisation compared to nonexendin-4 ligands (35). As GLP-1R dimerisation is required for full signalling efficacy (36), it is possible that this phenomenon partly explains our results.…”

The Influence of Peptide Context on Signaling and Trafficking of Glucagon-like Peptide-1 Receptor Biased Agonists

“…It should be noted that, once occupancy differences are taken into account, exendin-4-like ligands show a comparative signaling deficit, as greater affinity did not fully translate into commensurate increases in signaling potency. A recent report describes the detection of two separate binding sites for exendin-4 on the GLP-1R extracellular domain (ECD), potentially in keeping with previous photoaffinity cross-linking data and apparently responsible for impaired receptor oligomerization compared to nonexendin-4 ligands . As GLP-1R dimerization is required for full signaling efficacy, it is possible that this phenomenon partly explains our results.…”

A dimerization-dependent mechanism regulates enzymatic activation and nuclear entry of PLK1

“…While FRET signals were not detected in control cells (CFP, YFP, or CFP/YFP), we found a 10–14-fold increase in FRET signals in cells expressing C-CFP-PLK1 and C-YFP- PLK1 or C-YFP-PLK1 and N-CFP-PLK1, supporting the oligomerization of PLK1 in living cells. Next, we applied the NanoBit method [ 36 ] and expressed one or two PLK1 variants fused at the N- or C-terminal ends to LgBiT or SmBit. The luminescence originating from protein-protein interactions that reconstitute the complete luciferase complex was determined (Fig.…”