Signaling Pathway Mediated By 5-HT1AR/OX1R Heterodimers And Their Cellular Function

Orexin receptor 1（OX1R）and serotonin 1A receptor（5-HT1AR）,both of which belong to G protein-coupled receptors,play an important role in pathogenesis of depression.More and more researches have shown that most of the GPCRs can form dimers.However,no studies have been reported about the formation of5-HT1AR/OX1R dimerization.In this study,we reported the formation of 5-HT1AR/OX1R heterodimers and examined the signaling pathway mediated by the dimerization complexes.We further investigated the function of the dimer in pathological processes of depression.The present study demonstrated that 5-HT1AR was colocalized with OX1R on cell membrane by immunofluorescence.And the PLA results revealed the formation of 5-HT1AR and OX1R dimer in vitro and in vivo.Mass spectrometry analyses indicated the critical interface of 5-HT1AR/OX1R dimerization.To explore the signaling pathway caused by the heterodimer of the two receptors,we transfected into human embryonic kidney neoplasm（HEK293T）with 5-HT1AR,OX1R or the two receptors in combination.The effect of the dimers on the G_q signaling pathway was detected by Ca²⁺kit（Fluo-4-NW）.The level of cyclic adenosine monophosphate（AMP）was evaluated by BRET.Dual-luciferase assay was used to test the expression of nuclear factor-activated T cells（NFAT）,c AMP response element（CRE）etc.The binding efficiency of 5-HT1AR/OX1R dimer to G_q,G_s or G_i proteins was analyzed by BRET.Western blotting was used to determine the level of the c AMP response element binding protein（CREB）.Finally,we explored the function 5-HT1AR/OX1R heterodimers in chronic unpredictable mild stress（CUMS）depression model by immunofluorescence and PLA.The present study confirmed the expression of both 5-HT1AR and OX1R on the cell membrane and the dimers formation of the two recptors in vitro and in vivo.The results of mass spectrometry showed that TMD4,5 were responsible for the binding of 5-HT1AR/OX1R dimers in inactivated state,however,the interface was transformed from TMD4,5 into TMD6 in activation state.After administration of the appropriate agonist,5-HT1AR/OX1R heterodimers reduced the intracellular Ca²⁺concentration significantly.And the levels of c AMP significantly increased compared with the original monomers（p<0.05）.Co-stimulation of Orexin A and 8-OH-DPAT resulted in the upregulation of CRE of HEK293T-5-HT1AR/OX1R group（p<0.01）,while the expression levels of NFAT and SRE signal factor were significantly reduced（p<0.001）,indicating that the heterodimer of 5-HT1AR and OX1R preferred to bind G_s protein pathway.The binding ability of HEK293T-5-HT1AR/OX1R cells to G_qprotein following the treatment of Orexin A and 8-OH-DPAT was weaker than that of HEK293T-OX1R cells with Orexin A stimulation,The binding ability of HEK293T-5-HT1AR/OX1R cells to G_s protein after co-activation of Orexin A and8-OH-DPAT was significantly higher than that HEK293T-OX1R cells stimulated by Orexin A and HEK293T-5-HT1AR cells stimulated by 8-OH-DPAT（p<0.01）,these results suggested that the 5-HT1AR/OX1R heterodimer was biased toward to the G_ssignaling pathway.Western blotting resulted:p-CREB level of 5-HT1AR/OX1R group was significantly increased after orexin A and 8-OH-DPAT stimulation compared with 8-OH-DPAT-stimulated 5-HT1AR group and orexin A-stimulated OX1R group,in 5-HT1AR/OX1R group,the expression level of p-CREB reached a peak at 10 minutes following agonist stimulation（p<0.01）,we detected the expression level of p-CREB after stimulation using differnent concentration of Orexin A or8-OH-DPAT（0-100 n M）We found that the expression of p-CREB in HEK293T-5-HT1AR/OX1R group significantly increased at 100 n M concentration of the agonist（p<0.001）.In the PKA inhibitor H89 treatment group,the p-CREB level of5-HT1AR/OX1R dimer was significantly lower than the untreated group（p<0.01,p<0.05）,whereas in the PLC inhibitor U73122 treatment group,the p-CREB level of5-HT1AR/OX1R dimer was comparable to that of the unstimulated group,showing that 5-HT1AR/OX1R dimer affected CREB phosphorylation mainly through the c AMP/PKA signaling pathway rather than the PLC/PKC pathway.We built the animal model of depression by CUMS method,behavioral experiments included the syrup preference test（p<0.001）and weight gain experiment（p<0.01）were performed to test the efficiency of rat depression model.We detected the express pattern of5-HT1AR and OX1R in rat brain slices by immunofluorescence.PLA method was used to investigate the changes of 5-HT1AR/OX1R dimers in hippocampus of depression group compared to the control rat.The results showed that5-HT1AR/OX1R dimer levels were significantly decreased than that of the control group,while treatment of orexin A and 8-OH-DPAT improved the levels of5-HT1AR/OX1R dimmers,indicating the important role of the 5-HT1AR/OX1R dimers in depression.The present study demonstrated that 5-HT1AR and OX1R could form heterodimers and 5-HT1AR/OX1R changed the signaling pathway of the original monomeric caused by the agonists Orexin A and 8-OH-DPAT.The 5-HT1AR/OX1R preferred to bind G_s protein rather than G_q or G_i protein,and it explained the formation of 5-HT1AR/OX1R dimer and its influenced on the downstream signaling pathway;Furhtermore,our data showed that the levels of 5-HT1AR/OX1R dimer affected the development of depression,and also provided an important theoretical basis for the clinical treatment of depression.