| Objective: PAC1-R is a specific receptor of pituitary adenylate cyclase activating polypeptide(PACAP),which mediates significant anti-oxidation,anti-inflammatory,anti-apoptotic and cytoprotective functions of PACAP.Previous studies have found that: 1)SPAM1 and TAT can bind the allosteric regulatory site of PAC1-R in its extracellular domain PAC1-EC1 mimicing PACAP(28-38).SPAM1 and TAT can induce PAC1-R nuclear translocation.2)SPAM1 and TAT could effectively up-regulated the expression of PACAP.This study intends to explore the relationship between the nuclear translocation of PAC1-R induced by the PAC1-R allosteric regulation and the up-regulation of PACAP expression.The aim is to verify the mechanism by which the nuclear translocation of PAC1-R induced by its allosteric regulation mediates the PACAP up-regulation and try to explore the details of this mechanism.Methods:(1)In this study,the binding sites of SPAM1 and TAT binding to PAC1-R were analyzed by computer molecular docking,and their affinity with PAC1-EC1 was detected by ITC.(2)Using bioinformatics method to obtain 2526 bp promote sequence in the-2500/+26 region of the human PACAP gene ADCYAP1,and then the promoter fragment was synthesized and cloned into a new promoter reporting vector pYr-Prom Detect.The PACAP promoter dual luciferase reporter vector pYr-Prom Detect-PACAP was obtained.And the activity of pYrProm Detect-PACAP was detecting in mouse retinal ganglion cells(RGC-5)and hamster ovary cells(CHO).(3)With PAC1-e GFP-CHO cells expressing PAC1-R tagged at C-terminus with e GFP(Enhanced Green Fluorescence Protein,e GFP),and RGC-5 cells expressing PAC1-R naturally,the nuclear translocation of PAC1-R induced by SPAM1,TAT and PACAP(28-38)was detected,and the effects of SPAM1,TAT and PACAP(28-38)on PACAP promoter activity and PACAP expression were also detected.The correction was analyzed.(4)The effects of N-acetylcysteine(NAC)and 2-bromohexadecane(2-BP)on PAC1-R nuclear translocation,and their effects on PACAP promoter activity and PACAP expression was explored.The correction was analyzed.(5)ChIP-PCR was used to confirm the binding of the C-terminus of PAC1-R with the related DNA sequence of PACAP promoter in the nucleus.Results:(1)The results of computer docking showed that SPAM1 and TAT mimic PACAP(28-38)to bind the allosteric regulatory site of PAC1-R in extracellular domain PAC1-EC1.ITC results showed that the affinity of SPAM1 and TAT with PAC1-EC1 was similar to PACAP(28-38)(2)Based on the bioinformatics related to PACAP promoter,we constructed a PACAP promoter dual luciferase reporter vector pYr-Prom Detect-PACAP.The activity of pYrProm Detect-PACAP tested in RGC-5 cells and CHO cells confirmed the successful construction of effective PACAP promoter activity reporter system.(3)SPAM1(1 u M),TAT(1 u M)and PACAP(28-38)(1 u M)induce the up-regulation of PAC1-R signal in the nucleus.It was suggested that SPAM1,TAT and PACAP(28-38)can induce PAC1-R nuclear translocation.(4)SPAM1(0.01 u M ? 100 u M),TAT(0.01 u M ? 100 u M)and PACAP(28-38)(0.01 u M ?100 u M)effectively activate PACAP promoter activity,and the activation effect was stronger at low concentration(?1 u M),while the activation effect was weakened with the increase of concentration.The results of PACAP promoter were consistent with the expression trend of PACAP protein detected by ELISA.It was suggested that SPAM1,TAT and PACAP(28-38)can effectively up-regulate the activity of PACAP promoter and PACAP expression.(5)The up-regulation of PAC1-R signal in nucleus was inhibited by NAC or 2-BP,indicating that PAC1-R nuclear translocation induced by PAC1-R allosteric regulation can be inhibited by NAC or 2-BP.Meanwhile,NAC and 2-BP effectively inhibited the up-regulation of PACAP promoter activity and PACAP expression induced by SPAM1,TAT and PACAP(28-38).These results suggested that there was a positive correlation between PAC1-R nuclear translocation induced by SPAM1,TAT and PACAP(28-38)and the up-regulation of PACAP expression.(6)After the nuclear translocation of PAC1-R induced by SPAM1,the antibody raised against C-terminal of PAC1-R was used in ChIP-PCR,and the positive target bands were amplified by the primers targeting PACAP promoter-related sequences.It was suggested that the C-terminal of PAC1-R can bind to PACAP promoter-related sequences after entering the nucleus,and regulate promoter activity of PACAP.Conclusion: This study verified the existence of the mechanism by which PAC1-R nuclear translocation induced by PAC1-R allosteric regulation mediates the up-regulated expression of its specific ligand PACAP.In detail,SPAM1,TAT and PACAP(28-38)induce PAC1-R nuclear translocation through allosteric regulation,and nuclear PAC1-R regulates the activity of PACAP promoter through its C-terminal and up-regulates PACAP expression in positive correlation way. |
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