Study On The Mechanism Of Heterodimerization Of Apelin Receptor And Orphanin Peptide Receptor

G protein-coupled receptors(Gprotein-coupled receptors,GPCRs)dimers and oligomers are currently the focus of GPCRs family research.Preliminary studies have shown that GPCRs dimers not only exist as constitutive forms,but also form inducible dimers under the action of different ligands.GPCRs dimers not only interact with each other in the spatial structure to constrain each other,but also affect or alter the intrinsic efficacy and function of another receptor.Studying the molecular mechanism of dimer formation has important theoretical basis for elucidating the underlying signaling pathway and physiological functions.The putative receptor protein related to AT1(APJ)is belongs to the GPCRs of the class A mahogany-like family.Recent studies have shown that Apelin receptors play a significant protective role in cerebral ischemia,Parkinson's disease and Alzheimer's disease.Studies have shown that Apelin receptors can form homodimers or heterodimers with various receptors such as APJ-KOR dimer and so on.The opioid receptor-like 1 receptor(ORL1)also belong to the class A rhodopsin-like receptor family and play an important role in diseases such as addiction,depression and pain.Based on the previous research,this experiment attempts to explore the interaction between APJ and ORL,and the key sites of interaction,and explore the impact of the two on the downstream signaling pathway,and hope to provide a theoretical basis for further understanding of the molecular mechanism of dimer formation.The present study would provide scientific ideas for explaining the spatial conformation of dimers.In present study,we first detected the expression of APJ and ORL1 in SH-SY5 Y cells,and analyzed the subcellular distribution of APJ and ORL1 by laser scanning confocal microscopy.Then,we used technologies of the bioluminescence resonance energy transfer technology(BRET),fluorescence resonance energy transfer technology(FRET),Co-Immunoprecipitation(Co-IP)and Proximity ligation assay(PLA)to verify whether APJ and ORL1 can form dimers.After confirming that the two can occur heterodimerization,further explore the influence of agonis intervention,dimerization on receptor signal transduction.The e BRET assay was used to detect the coupling of G protein and the level of c AMP,the change of calcium ion was detected by FLuo-4,the expression of downstream signal element SRE and NFAT was detected by double fluorescent reporter gene method,the expression of p-CREB expression changes.At the same time,the interaction between dimerized APJ or ORL1 and ?-arrestern1 / 2 was detected by e BRET to further explore the possible influence of dimerization on the intracellular transport of the receptor.In addition,the key sites of heterodimerization of APJ and ORL1 were studied by mass spectrometry.The experimental results showed that:(1)The plasmid for the experiment was successfully constructed and sequenced correctly,which could be used in the next experiment.(2)Co-expression of APJ and ORL1 in SH-SY5 Y cells.Confocal laser scanning showed that APJ and ORL1 were highly expressed on the cell membrane.(3)The BRET,FRET,Co-IP and PLA technologies all confirmed the existence of heterodimerization interaction between APJ and ORL1.(4)After dimerization and binding of ORL1,the coupling activity of APJ with G?i1 increased and the coupling(p<0.05)with G?q decreased(p<0.05).Compared with the APJ independent expression group,the intracellular calcium content of APJ and ORL1 co-expression group after Apelin13 stimulation Decreased c AMP level,downstream signal element SRE content increased(p<0.01),NFAT content decreased,(p<0.05)c AMP response element(CREB)phosphorylation level decreased(p<0.05).(5)After dimerization and binding of ORL1,the interaction between APJ and ?-arrestern1 / 2 was also inhibited with an inhibition rate of 40%(p<0.05).(6)The results of mass spectrometry showed that the TM1 and TM5 transmembrane peptides are the key peptides for heterodimerization of APJ and ORL1.We constructed and screened the mutants and found that ORL1-L57 A,ORL1-L229 A,APJ-M36 A,APJ-V80 A,APJ-L218 A,APJ-I224 A play a key role in heterodimerization of APJ and ORL1.In conclusion,we think there is heterodimerization interaction between APJ and ORL1,and this dimerization can not only inhibit the internalization of APJ,but also promote the activation of Gi signaling pathway.However,TM1,TM5 The transmembrane region is the key peptide for heterodimerization of APJ and ORL1.