Institute of Neurobiology, School of Mental Health, Jining Medical University, Jining 272067, PR China.
Department of Nephrology, Zoucheng People's Hospital, Zoucheng 273500, China.
Cell Signal. 2020 Sep;73:109671. doi: 10.1016/j.cellsig.2020.109671. Epub 2020 May 12.
Apelin receptor (APJ) and bradykinin B2 receptor (B2R) play an important role in many physiological processes and share multiple similar characteristics in distribution and functions in the cardiovascular system. We first identified the endogenous expression of APJ and B2R in human umbilical vein endothelial cells (HUVECs) and their co-localization on human embryonic kidney (HEK) 293 cells membrane. A suite of bioluminescence and fluorescence resonance energy transfer (BRET and FRET), proximity ligation assay (PLA), and co-immunoprecipitation (Co-IP) was exploited to demonstrate formation of functional APJ and B2R heterodimer in HUVECs and transfected cells. Stimulation with apelin-13 and bradykinin (BK) increased the phosphorylation of the endothelial nitric oxide synthase (eNOS) in HUVECs, which could be inhibited by the silencing of APJ or B2R, indicating the APJ-B2R dimer is critical for eNOS phosphorylation in HUVECs. Furthermore, the increase of NOS and extracellular signal regulated kinases1/2 (ERK1/2) phosphorylation mediated by APJ/B2R dimer can be inhibited by U0126 and U73122, respectively, suggesting that the heterodimer might activate the PLC/ERK1/2/eNOS signaling pathway, and finally leading to a significant increase in cell proliferation. Thus, we uncovered for the first time the existence of APJ-B2R heterodimer and provided a promising new target in cardiovascular therapeutics.
Apelin 受体 (APJ) 和缓激肽 B2 受体 (B2R) 在许多生理过程中发挥重要作用，在心血管系统中的分布和功能上具有多个相似特征。我们首先鉴定了人脐静脉内皮细胞 (HUVEC) 中 APJ 和 B2R 的内源性表达及其在人胚肾 (HEK) 293 细胞膜上的共定位。我们使用一系列生物发光和荧光共振能量转移 (BRET 和 FRET)、邻近连接分析 (PLA) 和共免疫沉淀 (Co-IP) 来证明功能性 APJ 和 B2R 异二聚体在 HUVEC 和转染细胞中的形成。用 apelin-13 和缓激肽 (BK) 刺激可增加 HUVEC 内皮型一氧化氮合酶 (eNOS) 的磷酸化，APJ 或 B2R 的沉默可抑制其磷酸化，表明 APJ-B2R 二聚体对 HUVEC 中 eNOS 磷酸化至关重要。此外，APJ/B2R 二聚体介导的 NOS 和细胞外信号调节激酶 1/2 (ERK1/2) 磷酸化的增加可分别被 U0126 和 U73122 抑制，表明该异二聚体可能激活 PLC/ERK1/2/eNOS 信号通路，最终导致细胞增殖显著增加。因此，我们首次揭示了 APJ-B2R 异二聚体的存在，并为心血管治疗提供了一个有前途的新靶点。
