胰高血糖素样肽-1受体(Glucagon-like peptide-1 receptor, GLP-1R)属于B族G蛋白偶联受体(G-protein coupled receptor, GPCR)，经相关配体刺激后，可葡萄糖依赖地调节胰岛素分泌，是治疗2型糖尿病的重要靶点。对GLP-1R特异性配体的高通量筛选与结合特征评价，在发现候选靶向药物过程中具有重要意义。GLP-1R可发生同源/异源二聚；探究其二聚体在下游信号转导过程中发挥的作用，是开发变构调节剂及双重/多重激动剂的基础。

  本研究基于NanoBiT (NanoLuc Binary Technology)荧光素酶片段互补化学发光检测，构建了一种灵敏度高、简便快捷、经济性强的体外无细胞配体-受体结合体系，以便对GLP-1R与其相关配体间的结合特征及有关参数进行评价。由于首次将NanoBiT体系应用于体外评价配体-受体结合特征，本研究通过添加BSA及降低底物工作浓度等方式，对建立的NanoBiT检测体系进行优化。利用该体系测得荧光素酶片段融合的GLP-1受体N端胞外段(N-terminal extracellular domain, NTD)与其相关配体结合亲和力，与用经典方法测定的天然GLP-1R NTD数据大致相符。

  研究过程中发现GLP-1R NTD与相关配体间的结合存在负性协同作用，在配体加速解离实验与Scatchard分析中均得以证实，提示NTD二聚体的存在。NanoBiT体系化学发光检测的结果表明，独立的GLP-1R NTD在体外存在二聚/寡聚现象；该结论通过经典的分析型超离心法确证，测得其单体二聚化K_d值为4.5 µM，单体三聚化K_d值为26.9 µM，与在NanoBiT体系中仅考虑受体二聚化所测得的9.8 µM基本相符。通过对全长GLP-1R第4跨膜区(transmembrane 4, TM4)进行定点突变(G252A，L256A，V259A)，破坏经TM4介导的GLP-1R二聚体，在细胞水平确认NTD也参与了GLP-1R二聚体/寡聚体的形成。经实验验证，不论是体外NTD二聚体、细胞水平野生型GLP-1R二聚体，还是TM4突变型GLP-1R二聚体形成，均可被高浓度艾塞那肽(exenatide)抑制；但未观察到GLP-1(7-36)NH₂及杜拉鲁肽(dulaglutide)的抑制作用。

  为充分考虑反应体系中各个分子间相互作用关系，本研究创新性地建立一种基于反应规则的拟合模型。利用不同数学模型，对NTD结合GLP-1(7-36, A8G)探针的相关参数进行分析，经1:1结合模型拟合所得K_d值与纳入受体二聚化过程的分子结合规则模型分析结果近似(6~8µM)。竞争结合实验中，与GLP-1(7-36)NH₂和 dulaglutide相比，exenatide同GLP-1R NTD结合的亲和力更强，与报道中全长GLP-‍1受体的亲和力接近，并且显示出独特的双相结合特征，这在先前exenatide与GLP-‍1R或NTD的结合研究中从未报道过，充分体现了NanoBiT化学发光检测系统的高精度与高灵敏度。经双位点竞争模型分析，拟合得到exenatide结合NTD的两个K_i值，分别为1.4 pM和8.7 nM。本研究首次报道了GLP-1(7-37, A8G)二价激动剂dulaglutide与NTD的亲和力参数；经热力学竞争实验测得其与无His标签融合NTD探针(R-‍SmBiT)结合时的解离常数为22.5 nM，亲和力较天然GLP-‍1升高接近2个数量级。

  进一步对GLP-1R二聚体及单体形式介导下游信号转导特征的异同展开研究。通过构建NanoLuc荧光素酶大片段(LgBiT)融合的几种mini G蛋白探针与β-arrestin2探针，利用NanoBiT化学发光检测研究GLP-1受体对下游信号分子的募集；发现相比于GLP-1R，TM4突变的GLP-1R对mini G蛋白及β-arrestin2探针的募集效能(E_max)显著降低，而效价(EC₅₀)未有明显变化，这与文献及本研究中检测cAMP生成信号时观察到的现象恰好相反。这种信号分子募集特征变化与cAMP生成信号特征变化的截然不同，可能是信号转导过程中特定机制决定的普遍规律。在研究GLP-‍1R募集β-arrestin2的时相特征时，发现β-arrestin2探针募集的时程曲线呈单峰状，这可能是β-arrestin2与GLP-1R间发生短时相互作用的表现；受体突变后，β-arrestin2募集的时程曲线的峰型更加尖锐。

  综上所述，本研究基于NanoBiT建立了一种GLP-1R与相关配体结合特征的评价体系，可简便高效地在体外进行筛选，以发现候选GLP-1R靶向药物。在分子及细胞水平均证实了胞外段参与GLP-1R的二聚/寡聚作用，该结论为进一步开展受体胞外段是否参与其他B族GPCRs聚合的研究提供依据。同时，本研究利用NanoBiT对GLP-1R聚合形式在下游信号转导过程中的影响展开初步探索，为未来GLP-1R同源/异源二聚体下游信号的研究以及双靶点/多靶点激动剂类降糖药物的开发提供新思路。

  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 (K_d) derived from the ultracentrifugation analysis for the GLP-1R NTD dimerization was 4.5‍ ‍µM with that 26.9 µM for trimerization, which is comparable to the parameter (9.8 µM) 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 K_d for the probe GLP-1(7-36, A8G) is similar (6~8 µM) 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 K_i 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 molecules' recruitment to GLP-1R by constructing the LgBiT fused mini G probes and β-arrestin2 probe. It was found that, compared with the wild-type GLP-1R, the efficacy (E_max) of the mini G probes or β-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 β-arrestin2 probe recruitment to GLP-1R, probably attributing to a short interaction time between the two molecules. The peak turned sharper for the β-‍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-1R 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.