Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway

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.