Reassessment Of GLP-1R Ligand Binding, Self-aggregation, And Downstream Signaling Characteristics

The glucagon-like peptide-1 receptor(GLP-1R)belongs to the family B G-protein coupled receptor(GPCR).It could regulate insulin secretion in a glucose-dependent way following stimulation by ligands and is thus an important therapeutic target for type 2 diabetes.The candidate agonists to GLP-1R discovered from the high-throughput screening based on the binding characteristics detection are significant for the relevant new drug development.The homodimerization/heterodimerization of GLP-1R may be functional in the signal transduction,but their detailed roles remain unclear.Exploration of the specific roles of the GLP-1R dimers in signal transduction would be the foundation for the further development of the allosteric regulators and dual/multi-target agonists.A homogeneous ligand-receptor binding analysis system was constructed in vitro,applying a novel NanoLuc Binary Technology(NanoBiT)based on the luminescence detection of the split luciferase fragments complementation.The NanoBiT system was optimized for the ligand-receptor binding detection by adding BSA and decreasing the substrate concentration.The optimized NanoBiT system was successfully applied to evaluate the binding characteristics of the N-terminal extracellular domain(NTD)of GLP-1R with its related ligands,getting comparable results to other classic methods.The negative cooperativity of the ligands binding on the NTD of GLP-1R was observed,which was confirmed by the accelerated dissociation and Scatchard analysis.The negative cooperativity of the ligands binding implied the possible dimerization of the NTD.The dimerization/oligomerization of the isolated NTD was observed by NanoBiT directly and validated by the classic analytical ultracentrifugation.The dissociation rate constant(Kd)derived from the ultracentrifugation analysis for the GLP-1R NTD dimerization was 4.5 ?M with that 26.9 ?M for trimerization,which is comparable to the parameter(9.8 ?M)derived from the NanoBiT detection ignoring the NTD oligomerization.The possible involvement of the NTD in the full-length GLP-1R dimerization/oligomerization was also validated at the cellular level,with the significant luminescence remanent for the transmembrane domain 4(TM4)mutated(G252A,L256A,V259A)GLP-1R probes.It was discovered that exenatide under high concentration damaged the receptor dimerization,no matter for the NTD,the wild-type GLP-1R,or the TM4 mutated GLP-1R.Given the complex interaction between molecules in the reaction system,the study developed a novel model based on the binding rules.In an analysis of the parameters of the NTD binding with different models,the Kd for the probe GLP-1(7-36,A8G)is similar(6?8 ?M)in both the 1:1 binding model and the receptor dimerization model.For the competition experiment,exenatide showed a much higher binding affinity to the GLP-1R NTD compared with GLP-1(7-36)NH2 and dulaglutide,which was close to that affinity with the full-length GLP-1R.Specially,exenatide displayed a two-site binding character,with the fitted Ki values of 1.4 pM and 8.7 nM for the two sites,respectively.To our best knowledge,the two-phase competition curve of exenatide binding to GLP-1R or NTD was not reported before.Our study also reported the dissociation constant of 22.5 nM for NTD probe(R-SmBiT)binding with the bivalent GLP-1(7-37,A8G)dulaglutide for the first time.This affinity is about 2 orders higher than that for the native GLP-1.The study further investigated the difference between the signal transduction characteristics mediated by GLP-1R dimer and monomer.The NanoBiT system was applied for the analysis of the signaling mo lecules' recruitment to GLP-1R by constructing the LgBiT fused mini G probes and ?-arrestin2 probe.It was found that,compared with the wild-type GLP-1 R,the efficacy(Emax)of the mini G probes or ?-arrestin2 probe recruitment to the TM4 mutated GLP-1R was reduced substantially,while the relevant potency(EC50)remained.This was just reverse to the parameter variation characteristics of the cAMP accumulation in the reference and our present study.The distinct difference of the characteristics between the signaling molecules recruitment and the cAMP accumulation may be a rule in the signal transduction,which is possibly determined by specific mechanisms.A single peak rather than a platform was observed for the time-course curve of the ?-arrestin2 probe recruitment to GLP-1R,probably attributing to a short interaction time between the two molecules.The peak turned sharper for the ?-arrestin2 probe recruitment to the TM4 mutated GLP-1R.In conclusion,our study constructed an efficient platform for the evaluation of ligands binding characteristics with GLP-1R based on the NanoBiT.This platform could contribute to the discovery of candidate drugs from the effective screening of the ligands.The study confirmed the involvement of the NTD in the GLP-1 R dimerization/oligomerization at both the molecular and the cellular level,which suggests the possible roles of the NTD in other family B GPCRs dimerization.At the same time,the importance of the GLP-1R dimerization in signal transduction was preliminarily researched by the NanoBiT,providing new ideas for the further investigation of the GLP-1R homodimerization/heterodimerization and development of the novel multi-target drugs.