Heterodimerization Of Apelin Receptoe And Neurotensin Receptor1in Living Cell

ObjectiveTo explore the possibility that Neurotensin receptor1（NTR1） and Apelin receptor（APJ） can form heterodimer with novel signal transduction characteristics, and to providehelpful data for understanding physiological function and pathophysiological mechanismthat they envolved in and provide new target for drug development.Methods1.Human umbilical vein endothelial cells （HUVECs） culture: HUVECs were culturedin RPMI-1640medium supplemented with10%fetal calf serum at37℃in an atmosphereof5%CO2.2.The endogenous expression of NTR1and APJ in HUVECs: RNA and proteins ofHUVECs were collected in order to detect the endogenous expression of NTR1and APJwith Reverse transcription-polymerase chain reaction （RT-PCR） and Western blot,respectively.3.Construction of eukaryotic expression vectors and its expression in HEK293cells:eukaryotic expression vector containing full length cDNA of human NTR1or APJ wasconstructed for BRET and FRET detection. In BRET, human NTR1was tagged by Renillaluciferase （Rluc）（pRluc-hNTR1-pcDNA3.1） which was used as donor, and human APJwas tagged by enhanced green fluorescence protein（EGFP）（pEGFP-hAPJ-pcDNA3.1）used as acceptor plasmid. In FRET, human NTR1was tagged by cyan fluorescent protein（CFP）（pCFP-hNTR1-pcDNA3.1） which was used as donor, and human APJ was taggedby yellow fluorescence protein（YFP）（pYFP-hAPJ-pcDNA3.1）used as acceptor plasmid.Then, pRluc-hNTR1-pcDNA3.1was identified by confocal scanning microscope laser andWestern blotting.4.Co-localization was carried out on APJ/NTR1cotransfected HEK293cells withconfocal scanning microscope laser. 5.A novel technique bioluminescence resonance energy transfer （BRET） was carriedout to explore the heterodimerization between NTR1and APJ in living cells. Saturationassays, several samples that coexpress a constant amount of donor-labeled protein withincreasing amounts of acceptor-labeled protein are assayed; ligand-induced assays, BRETratio could be detected with treatement of apelin-13or neurotensin （NT）.6.Another novel technique Fluorescence resonance energy transfer （FRET） wascarried out to further explore the heterodimerization between NTR1and APJ in living cells.Donor plasmid pCFP-hNTR1-pcDNA3.1and receptor plasmid pYFP-hAPJ-pcDNA3.1was cotransfected HEK293cells, then FRET signal was detected after12-24h.7.ERK1/2phosphorylation was detected by western blot analysis. In order to detectdose-dependent effect of ERK1/2phosphorylation, HEK293-APJ and HEK293-APJ/NTR1were treated with10-5-10-8mol/L apelin-13, respectively. HEK293-NTR1andHEK293-APJ/NTR1were treated with10-5-10-8mol/LNT, respectively.Results1.HUVECs grew fast, arranged in “cobblestone”-like pattern, and displayed themorphology of larger polygonal cells. No overlapping growth was observed in the culture.2.RT-PCR and Western blot suggested both NTR1and APJ express in HUVECs.3.Double digestion and the sequencing of the Shanghai Sangon suggested alleukaryotic expression vectors used for BRET and FRET were successfully constructed.Then, pRluc-hNTR1-pcDNA3.1was identified by confocal scanning microscope laser andWestern blotting, showing that it was localized on plasma membrane and this localizationwas consistent with its normal physiological localization.4.Co-localization was carried out on APJ/NTR1cotransfected HEK293cells, LSCMrevealed a high degree co-localization of APJ with NTR1, predominantly to the plasmamembrane, suggesting that NTR1and APJ can exis as heterodimers.5.Saturation assays suggested a constitutive interaction between NTR1and APJ.However, BRET signal increased with the treatement of apelin-13or neurotensin,suggesting a promoted affinity with their ligand.6.FRET further suggested NTR1and APJ can exis as heterodimers.Together,Co-localization, BRET and FRET assays confirmed heterodimerization between NTR1andAPJ.7.From10^-5to10^-8mol/L, ERK activation in HEK293-APJ/NTR1cells wassignificantly higher than that in HEK293-APJ or HEK293-NTR1cells. Intresetingly, theconcentration of NT inducing top ERK activation in HEK293-NTR1and HEK293- NTR1/APJ is10-7and10-8mol/L, respectively; the concentration of AP inducing top ERKactivation in HEK293-APJ and EK293-NTR1/APJ is10-6and10-7mol/L, respectively.ConclusionsThese combined results demonstrate that human APJ forms a heterodimer with NTR1or the first time, and the heterodimer can increase ERK phosphorylation. Heterodimerization between APJ and NTR1could involed in physiological function such as cardiovaclarprotection and neuroprotection, as well as pathophysiological mechanisms. And theseresults will help to retrieve new pharmacological targets for new drug development. Futurestudies will focus on the structure basis and physiological consequences of APJ andNTR1heterodimerization.