The Mechanism Of Interaction Between Apelin Receptor And β-arrestins

G protein-coupled receptors (GPCRs), known as seven transmembrane receptors(7-TMRs), are the largest protein receptor superfamily. These receptors and their ligandsdirect a diverse array of physiological and pathological responses. Half of drug targets in thepharmaceutical industry are GPCRs. β-arrestins (β-arrestin1and β-arrestin2)are main focusedreverse regulators in GPCRs signaling. As intracellular adaptor and scaffolding proteins,β-arrestins play important roles in GPCR desensitization, internalization, intracellulartrafficking and G-protein-independent signaling. Signaling differences beween correspondingligand stimulation of G protein signaling and (or) β-arrestins can be the final decision of theGPCRs ligand-specific cell biological effects. The interactions between G-protein-coupledreceptors and β-arrestins determine the selectivity and efficiency of receptor signaling. Basedon the interaction beween GPCRs and β-arrestin, GPCRs are divided into A and B classes.Class A GPCRs exhibiting weak, transient β-arrestin interactions with an apparent preferencefor β-arrestin2, however that form strong, stable interactions with both β-arrestin1andβ-arrestin2tending to be denoted class B receptors for β-arrestin usage.Recent developments in BRET provide biophysical insights into real-time and dynamicmonitoring of GPCR-β-arrestin complexes in living cells. In concert with other methods suchas confocal microscopy, co-Immunoprecipitation, assays for studying internalization andrecycling kinetics, now researchers can elucidate the ever-expanding roles of β-arrestins inmediating GPCRs functions.Apelin and its G protein-coupled receptor(APJ)regulate blood pressure, intake of foodand water, the release of hormones and neuropeptides and cardiac contractility as well as inthe modulation of immune function. So many physiological regulation functions involved inthe central nervous and cardiovascular systems functions making it one of promising drugtargets to treat hypertension, ischemic heart disease, heart failure in the future. However, it isnot known that which Class APJ belongs to A or B type GPCRs? It is also not clear that therole of β-arrestin in the regulation of APJ internalization, desensitization, endocytosis, resensitization and signal transduction.This study helped to understand the interaction between APJ and β-arrestins, and therelationship between structure and function of human APJ carboxyl-terminal tail to providenew target for new drug.To explore the interaction between APJ and β-arrestins, we constructed variousexpression vectors of APJ and β-arrestins, and transiently or stably transfeced into humanembryonic kidney293(HEK293) cells, and established stable cell lines that expressed APJ,HA-APJ. Based on the established cell lines, the interaction was detected by co-localization,Co-immunoprecipitation and BRET assays for real-time, dynamic monitor of GPCR-β-arrestin complexes in living cells for prolonged periods.HEK293cells alone or co-transfected with HA-APJ and eGFP-β-arrestin1or eGFP-β-arrestin2, were pretreated with different concentration of apelin-13for20minutes, and thencollected for immunofluorescence staining; cells at different time points were collected forimmunofluorescence staining, confocal microscopy showing that: In0~5minutes, the receptorand β-arrestins co-localization at the cell membrane, and both had intracellular translocationas time past within30min.HEK293cells were transfected with HA-APJ and flag-β-arrestin1or flag-β-arrestin2plasmids, then the extracted protein was used immunoprecipitation experiments with HAantibody, the results showed that,100KD,95KD bands were obtained. in co-transfected withHA-APJ and flag-β-arrestin1or flag-β-arrestin2, repectively. When treated with anti-flagantibody for immunoprecipitation, the results showed the same. Therefore, it is suggested thatAPJ mainly interacts with β-arrestin2.In this study, two groups of eukaryotic constructed plasmids were used: peGFP-APJ-pcDNA3.1and Rluc-β-arrestin1, peGFP-APJ-pcDNA3.1and Rluc-β-arrestin2. There plasm-ids were transiently transfected into HEK293cells. After48h, BRET1and eBRET were usedto mornitor real time, dynamic interaction between the two proteins within living cells. Theresults showed, APJ combined with β-arrestin1earlier than with β-arrestin2; the BRET valueincreased lower and decreased rapidly, the BRET value of APJ and the β-arrestin1reached thetop value in20minutes, while that of APJ and the β-arrestin1in25minutes reached themaximum value,then declined rapidly. Then we carried out Graph pad prism5.0software nonlinear regression analysis to calculate the maximum amount of half-EC50values were(3.8±1.2)×10-8M,(5.0±1.2)×10-8M, the difference was not significant.Together, immunoprecipitation positioning, co-immunoprecipitation and BRET experi-ments have confirmed that the interaction between apelin receptor and β-arrestins in line withthe typical characteristics of type A receptors.Studies have confirmed that the carboxy-terminal of GPCRs is the main sites of Gprotein-coupled receptor kinase (GRK) phosphorylation. serine/threonine residues are majorphosphorylation sites. In order to study APJ carboxy-terminal phosphorylation sites and therelationship between cell signal transduction, We detected the key intracellular signalingmolecules: extracellular signal-regulated kinase(Extracellular-Regulated Kinases1/2, ERK1/2), cyclic adenosine monophosphate enzyme (cycle AMP, cAMP), serum response element(Serum response element, SRE) and cAMP response element (cAMP response element,CRE).cAMP is generated when HEK293cells transfected with APJ mutant C320Z, A332Z,A344Z, Q352Z is stimulated by forskolin(FSK).Enzyme-linked immunoassay(ELISA)suggested apelin-13can inhibit the process.The results showed that the level of theFSK-induced cAMP production was significantly lower in the mutant C320Z, A332Z, A344Z,except for Q352Z.Time and Concentration-dependent ERK1/2phosphorylation analyzed by Western blot.The results showed that, compared with wild-type receptor, treatment with apelin-13HEK293cells stable expression mutant C320Z, A332Z, A344Z, Q352Z can reduced ERK1/2phospho-rylation, reached to peak in15minutes then returned to baseline in60minutes. As forconcentration-dependent ERK1/2phosphorylation, treated with apelin-13with differentconcentration from10-11M~10-5M, ERK1/2phosphorylation increases with the concentrationvalues increased, but the mutant C332Z, A344Z, Q352Z elevated value relative lower thanthat of the wild-type APJ, the difference was significant.The Double-Report gene Studies have shown that, apelin activates ERK of APJ andmutants by inhibiting the phosphorylation APJ through PKC pathway, while cAMP-depen-dent protein kinase A (protein kinase A, PKA) pathway is always dependent on the activa-tion of the cAMP pathway. We used the Promage’s Dual Reporter Gene Assay Kit to mornitor two downstream signal SRE and CRE. In stably transfected HEK293-APJ and themutant-HEK293cells transient transfection pSRE-Luc or pCRE-Luc plasmide. As a result,SRE activity enhanced and CRE reduced. Compared with wild-type APJ, increased SREactivity ratio of the mutant cells is lower than that of the wild-type and mutant A344Z, Q352Zsignificantly. PKC inhibitor BisⅡcompletely blocked SRE-Luc activity, indicating that stabletransfection of wild-type and mutant APJ in HEK293cells, apelin-13-mediated SRE depend-ent enhancement of PKC, but deletion of all the carboxyl end of the threonine residue havestill not completely block the activity of SRE.In conclusion，we utilized BRET technology combined with immune co-localization,immunoprecipitation conforming that APJ is a classic A-type GPCRs for first time; Thereceptor internalizes into cell with the clathrin protein; Mutation of the carboxyl-terminalamino acid of APJ results in the C-terminal part trunction effecst on signal transductionsignaling molecules ERK1/2, cAMP, SRE and CRE. C-terminal of APJ plays an importantrole in the signal transduction of the intracellular signaling. The Clusts of Ser/Thr between332Ala and320C are most important for APJ signaling. The research results may be helpfulfor further revealing intracellular molecular mechanisms in the cardiovascular system andcentral nerous system of Apelin/APJ system. It also provide new experimental evidence andhas important application value for providing a new idea of targetting Apelin/APJ system drugdevelopment...