As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain.

作为G蛋白偶联受体（GPCR），5-羟基色胺1A受体（5-HT1AR）和orexin受体2（OX2R）调节细胞下游分子的水平。不同GPCR的异二聚体在各种神经系统疾病中起重要作用。此外，5-HT1AR和OX2R参与神经系统疾病的发病机制，例如海马可塑性缺乏的抑郁症。然而，两种受体的直接相互作用仍然难以捉摸。在本研究中，我们首先证明了5-HT1AR和OX2R的异二聚体形成。通过cAMP（Epac）cAMP生物发光共振能量转移（BRET）生物传感器分析直接激活的交换蛋白显示，与5-HT1AR组相比，转染了这两种受体的HEK293T细胞中细胞cAMP的表达水平显着增加。另外，在激动剂处理后，与5-HT1AR和OX2R组共转染的HEK293T细胞中钙的细胞水平被强烈上调。此外，蛋白质印迹数据显示5-HT1AR和OX2R异二聚体降低了细胞外信号调节激酶（ERK）和cAMP反应元件结合蛋白（CREB）的磷酸化水平。这些结果不仅揭示了5-HT1AR和OX2R异源二聚体的形成，而且表明该异源二聚体影响了下游信号传导途径，这将为大脑中两种受体的功能提供新的见解。免疫印迹数据表明，5-HT1AR和OX2R异二聚体降低了细胞外信号调节激酶（ERK）和cAMP反应元件结合蛋白（CREB）的磷酸化水平。这些结果不仅揭示了5-HT1AR和OX2R异二聚体的形成，而且表明异二聚体影响了下游信号传导途径，这将为大脑中这两种受体的功能提供新的见解。免疫印迹数据表明，5-HT1AR和OX2R异二聚体降低了细胞外信号调节激酶（ERK）和cAMP反应元件结合蛋白（CREB）的磷酸化水平。这些结果不仅揭示了5-HT1AR和OX2R异二聚体的形成，而且表明异二聚体影响了下游信号传导途径，这将为大脑中这两种受体的功能提供新的见解。