The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.

G蛋白偶联受体（GPCR）二聚体界面在二聚体的形成和稳定中起重要作用。因此，鉴定潜在的受体-受体界面是研究GPCR的重要部分。已经采用了各种策略来研究GPCR二聚体界面并探索其功能意义，但是实验方法缺乏鲁棒性，并且计算费力。在此，我们报告了一种组合的优化实验和计算方法，用于识别和结构表征GPCR二聚体界面，并构建原子分辨率模型。使用包含人类免疫缺陷病毒反式转录激活因子（HIV-TAT）蛋白转导基序的跨膜结构域（TM）肽，基质辅助激光解吸串联飞行时间质谱（MALDITOF-MS），以及生物发光共振能量转移（BRET），我们成功鉴定了Apelin受体（APJ）/ Nociceptin受体1（ORL1）和APJ / Vasopressin受体2（V2R）异二聚体界面，以及两个介导二聚化的关键位点。该方法可以鉴定GPCR同二聚体和异二聚体的二聚体界面。