Signaling Pathway Mediated By KOR/B2R Heterodimers And Their Cellular Function

The ?-opioid receptor(KOR)and bradykinin B2 receptor(B2R)both belong to the G protein coupled receptor family.The two receptors and their ligand,known as dynorphin and bradykinin,are wildly distributed in the cardiovascular system and central nervous system(CNS),exhibiting important function in cardiovascular and neuroprotection.To this day,it is unknown whether KOR and B2 R could interaction.In the present study,the interaction between the KOR and B2 R was detected by biophysics methods.The effects of KOR/B2 R heterodimers on signal transduction and cell function were also researched,which clarified the mechanism of heterodimer on signal transduction and neuroprotection,providing novel ways of treatment for nervous disease and new target for new drug.In the present study,we establish cell lines stably expressing KOR,B2 R or KOR and B2 R using the plasmids pcDNA3.1-KOR and pcDNA3.1-B2 R.Immunofluorescence staining showed the co-localization of both KOR and B2 R on the surface of HEK293 cells transfected with pEGFP-B2 R and pcDNA3.1-KOR.RT-PCR and Western blot confirmed the co-expression of KOR and B2 R in SH-SY5 Y cells.The plasmids KOR-Rluc and B2R-mVenus were constructed and transfected into HEK293 cells.The BRET ratio in cells co-expressing KOR-Rluc and B2R-m Venus was significantly increased compared to the control groups,suggesting that the KOR and B2 R can constitutively interact.In BRET saturation assays,BRET signal for the transfer of energy between KOR-Rluc and B2R-Venus increased as a hyperbolic function of the concentration of the Venus-fusion construct added reaching an asymptote,whereas the control group exhibited a nonspecific linear relationship.The results of competitive BRET analysis suggest that BRET signal of KOR-Rluc/B2R-mVenus was decreased as the concentration of unlabled KOR or B2 R increased.These results indicated that the interaction between KOR and B2 R is specific not due to receptors overexpression and random collisions.FRET was performed to further explore the interaction of KOR and B2 R.Compared to control group,FRET signal was higher in KOR-CFP and B2R-mVenus co-transfected HEK293 cells.The fact that FRET signal could also be detected in CHO cells co-transfected with KOR-CFP and B2R-mVenus plasmids indicated that the FRET phenomenon lived in distinct cell lines.Proximity ligation assays(PLA)directly confirmed the receptor–receptor interactions.To investigate the effects of GPCR heterodimer on signalling pathways,cAMP,cAMP response element(CRE),serum response element(SRE),and cAMP-response element-binding protein(CREB)were detected.Dyn A(1-13)significantly enhanced cAMP accumulation in co-transfected cells but not in single-transfected cells.Interestingly,BK cannot activate cAMP signaling pathway through KOR/B2 R heteromers,suggesting that heteromerization leads to different affinities for ligands.To investigate the interaction between KOR/B2 R heterodimer and G protein subunits,we prepared the HEK293 cells transiently expressing KOR,B2 R,or KOR and B2 R,together with G?s-Rluc8,G?i2-Rluc8,or G?q-Rluc8 and unlabled ?1 and ?2 subunits.After Dyn A(1-13)stimulation,the BRET ratio between KOR-Venus and G?s-Rluc8 significantly increased,suggesting that heterodimer leads to G?s protein binding and activation.The activity of CRE and SRE in transfected HEK293 cells was detected,and the results showed that CRE-luc activity was significantly higher in HEK293-KOR/B2 R cells than in HEK293-KOR and HEK293-B2 R cells,whereas SRE-luc activity was decreased.Due to the CRE activity and SRE activity depend on PKA and PKC activities,we further confirmed that the KOR/B2 R heterodimer leads to an increase in G?s binding.In general,GPCRs were coupled with G?s to activate adenylate cyclase(AC),increasing the levels of CREB phosphorylation.To determine whether KOR/B2 R heterodimers affect CREB phosphorylation,the time-and dose-dependent effects of Dyn A(1-13)on CREB activation were investigated.The ability of KOR and/or B2 R to stimulate phosphorylation of CREB was assessed by treating cells with Dyn A(1-13)(10-7M)for 0-60 min.A robust increase(maximal at 5min)in CREB phosphorylation was observed in HEK293-KOR/B2 R cells,with a subsequent decline.In dose-dependent experiments,CREB activation increased with increasing concentrations of Dyn A(1-13)in HEK293-KOR/B2 R cells.Similarly,a dose-dependent effect on CREB activation was also observed in human SH-SY5 Y cells.To examine whether CREB phosphorylation is PKA and PKC dependent in HEK293-KOR/B2 R cells,we treated the cells with PKA inhibitor H89 or PLC inhibitor U73122 prior to Dyn A(1–13).The results showed that levels of CREB phosphorylation in HEK293-KOR/B2 R cells was significantly lower with H89 treatment but not with U73122,suggesting that the KOR/B2 R heterodimer signals through the G?s/cAMP/PKA/CREB pathway after stimulation by Dyn A(1-13)in HEK293-KOR/B2 R cells.To strengthen the role of KOR/B2 R heterodimer in CREB phosphorylation,we used KOR shRNA or B2 R shRNA in SH-SY5 Y cells.Western blot analyses showed that treatment with shRNA-KOR or shRNA-B2 R significantly blocked the expression of KOR or B2 R protein.In these cells,Dyn A(1-13)treatment leads to a significant reduction in CREB phosphorylation compared with the control shRNA.Interestingly,B2 R depletion also reduced the Dyn A(1-13)induced CREB phosphorylation,suggesting that KOR and B2 R formed a functional heterodimer which mediated the enhanced CREB phosphorylation upon treatment with KOR ligand Dyn A(1-13).The above results indicate that KOR and B2 R can form heterodimer.To further investigate the role of the dimer in cell function,we performed a cell viability assay.Treatment with Dyn A(1-13)significantly increased the proliferation of HEK293-KOR/B2 R cells,while the proliferation of HEK293-KOR and HEK293-B2 R cells was not affected.Corresponding to its effects on CREB phosphorylation,Dyn A(1-13)promoted the proliferation of human SH-SY5 Y cells.In conclusion,We demonstrate for the fist time that human KOR can form heterodimer with B2 R,which induced a novel signaling pathway.The novel signaling pathway G?s/cAMP/PKA enhanced the levels of phospho-CREB and increased the cell proliferation.The study explored the mechanism of KOR/B2 R heterodimer on signal transduction and neuroprotection,providing novel ways of treatment for nervous disease and new target for new drug.