Apelin Receptor Homodimers/oligomers And Its Novel G Protein-dependent Signaling

ObjectivesApelin receptor,also called putative receptor protein related to ATI(APJ),belongs to the family A of G-protein-coupled receptor(GPCR).The receptor is involved in regulatory pathways of the cardiovascular and nervous systems,and is an important pharmacotherapeutic target.Our previous work has revealed that APJ could form hetero-dimers with other GPCRs(Kappa opioid receptor,neurotensin receptor type 1 etc.),which had distinct functional characteristics.However,the APJ homodimers and oligomers,mobility,interfaces and function remains to be investigated.Here,we applied total internal reflection fluorescence microscope(TIRFM),matrix assisted laser desorption/ionization time-of-flight mass spectrometer(MALDI-TOF MS),resonance energy transfer,and Real-Time Cell Analyzer(RTCA)to measure APJ homodimers/oligomers,distribution,dynamic process,and detect the interfaces of APJ homodimer and novel functional characteristics.The results may serve as a unique mechanism of intracellular signal transduction for fine-tuning APJ-mediated physiological and pathological processes,and help to provide theoretical and experimental basis for exploring new pharmacological targets and new drugs.Methods1,Visualization and analysis of GFP and APJ-GFP with TIRFMGreen fluorescence protein(GFP)molecules and APJ-GFP on the cell surface were monitored with TIRFM,equipped with a 100×1.47 NA oil-immersion objective,with only 8%laser power for imaging events,and relatively short exposure times(three frames per second).The penetration depth of the evanescent field was?150 nm.The particles were analyzed and fitted to a Gaussian function with Origin8.0 software.2.Assessment of APJ homodimers/oligomers and its G protein-dependent signaling with co-immunoprecipitation,resonance energy transfer and bimolecular fluorescence complementation-bioluminescence resonance energy transferCo-immunoprecipitation(Co-IP),bioluminescence resonance energy transfer(BRET)and fluorescence resonance energy transfer(FRET)were used to detect the APJ homodimers,and the effect of[A1a13]apelin-13,apelin-12,13,15,17 or 36 on homodimers,respectively.Bimolecular fluorescence complementation(BiFC)-BRET was used to detect the APJ homooligomers,and the effect of[A1a13]apelin-13,apelin-12,13,15,17 or 36 on APJ homooligomers,respectively.Additionally,the novel signaling(G protein-dependent signaling)of APJ homodimers was also explored using BiFC-BRET.3.Detection of dynamics of APJ homodimers with proximity biotinylationProximity biotinylation was performed with a laser confocal scanning microscope(LCSM)equipped with a 60×oil-immersion objective lens(To confirm the formation of APJ homodimers).Flow cytometry was performed with a BD FACSCalibur flow cytometer(To detect the dynamics of APJ homodimers).Phycoerythrin(PE)was excited by 488 nm laser and emission was examined with a 575/24 nm filter.4.Recognization of APJ homodimer interfaces with MALDI-TOF MSWe utilized the transmembrane domain(TMD)of APJ and MALDI-TOF MS to confirm the APJ homodimers and examine the dimer interfaces.A series of APJ mutants(APJM361.40A,APJL401.44A,APJV732.48A,APJT983.44A,APJV1484.44A,APJL2185.55A and APJF3077 51A,etc.)were constructed to find the key site with BRET1 and TIRFM.5.Real-time monitoring of human umbilical vein endothelial cells proliferation with RTCARTCA was used to monitor the proliferation of human umbilical vein endothelial cells(HUVECs),in order to measure the effects of TMDs on the apelin-13-induced cellular responses in HUVECs as reported via a cell index.Results1.Visualization and analysis of GFP and APJ-GFP on the surface of CHO cellsGFP and APJ-GFP were detected and visualized by TIRFM(488 nm).The distribution of the intensity of GFP particles was analyzed and fitted to a Gaussian function with Origin8.0.The results indicated a predominant peak,and the average intensity of single GFP was 308.08±3.38(mean±SD)(n=321).Th distribution of the intensity of APJ-GFP particles in cells with less than 0.3 particle/?m2 was analyzed and fitted to a Gaussian function with Origin8.0.The intensities of the individual spots were fitted to three Gaussian distribution and the average intensities were 311.7± 12.37,610.48±5.85,and 896.87±21.86(mean±SD)(n=1087),respectively.Given the analysis of GFP,this revealed a mixed distribution of APJ monomers,homodimers,and oligomers,and the proportion of APJ monomers,homodimers and oligomer was?40%,?-6%and?24%,respectively.APJ monomers,homodimers,and oligomers were distinguished according to the smallest normalized intensity y=(x-xc)/w.2.Formation of APJ homodimers and oligomers,which was agonist or antagonist independentCo-IP analysis of HA-APJ and Myc-APJ in co-transfected CHO cells revealed that APJ could form homodimers.BRET1 were performed to confim APJ homodimers with the negative controls(mOX2?R-Rluc+mOX2aR-EGFP)(*P<0.05,n=4).FRET was also performed to provide further evidence for APJ homodimers.a notable FRET signal was detected and FRET effiency was 56%(**P<0.01,n=4),indicating that APJ existed as a homodimer.When cells co-expressing APJ-Rluc and APJ-EGFP were treated with an antagonist([A1a13]apelin-13)or agonist(apelin-12,13.15,17 or 36),BRET signals were not significantly different from those of unstimulated cells,indicating APJ homodimer formation was independent of antagonist or agonist.Additionally,BiFC-BRET assays were performed to detect APJ oligomerization in CHO cells.A high BRET ratio was detected,which demonstrated the existence of APJ oligomers(**P<0.01,n=4).Furthermore,when cells co-expressing these receptors(APJ-Rluc+APJ-VC155+APJ-VN173)were treated with an antagonist([A1a13]apelin-13)or an agonist(apelin-12,13,15,17 or 36),the BRET ratio was the same as that of unstimulated cells,indicating that APJ oligomer formation was independent of antagonist or agonist.3.APJ homodimer dynamics on cell membranesThe APJ biotinylation increased linearly within?15min and reached saturation after 15 minutes(1.805%,13.3%and 22.5%),which likely reflected the continuous association and dissociation of APJ in a monomer-dimer dynamic equilibrium.Single-molecule co-tracking of APJ homodimers were detected by TIRFM in CHO cells,the images indicated APJ existed in a dimeric form with short dimer lifetimes of-900 ms.4.TMD1,TMD2,TMD3 and TMD4 provided APJ homodimer interfacesThe effects of TMDs on APJ homodimerization were examined by MALDI-TOF MS,finding TMD1,TMD2,TMD3 and TMD4 provided the most notable APJ homodimer interfaces.Furthermore,TMD1,TMD2,TMD3,and TMD4 signifiantly reduced BRET signals of APJ homodimers by 55-75%with BRET1(**P<0.01,n=4),indicating TMD11 TMD2,TMD3 and TMD4 were involved in dimer interfaces.5.Novel functional characteristics of APJ homodimersG?-Rluc8,APJ-VN173,and APJ-VC 155 were transfected into CHO cells and BRET signals were measured after treatment with apelin-13.The results indicated that APJ homodimers activated only Gai3 and G?q(APJ monomers activated G?i1,G?i2,G?i3,G?q,G?13,and G?0)(*P<0.01,n=4).We also measured the effects of APJ homodimers on apelin-13-induced cellular responses in HUVECs with RTCA.Co-stimulation apelin-13 with TMD1,which was demonstrated to disrupt APJ homodimers,led to a decreasing trend in the cell index(CI).Conclusions(1)APJ formed homodimers and oligomers.In cells with<0.3 particle/?m2,approximately 40%were identified as monomers,while 36%were homodimers,and 24%were oligomers.APJ homodimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of new receptor complexes.(2)TMD1,TMD2,TMD3 and TMD4 were involved in APJ homodimer interfaces,(3)APJ homodimers had distinct G protein binding profiles and cellular responses.APJ homodimers led to an increasing trend in the Cl via G?o3 and G?q.This research elucidates APJ dimerization and oligomerization,distribution,dynamic process,interfaces and ftunction.It has important theoretical significance for the study of chemical properties and intracellular signal transduction pathways of APJ dimer and oligomer,and provides detailed experimental data for the search of effective therapeutic targets and the development of new therapeutic targets,which has important application value.