Exploration Of Nuclear Translocation Of Neuropeptide Receptor PAC1-R Cells And Its Biological Role

Objective: More and more studies have shown that G-protein coupled receptors(GPCRs)occurs nuclear translocation,the phenomenon of which is that the receptors originally located on the cell membrane could be further localized on the nuclear membrane after internalization or translocation into the nucleus,mediating signal transduction pathways different from those on cell membranes.PAC1-R is the specific receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide(PACAP).Researches have shown that PAC1-R could further localize on nuclear membrane or enter into the nucleus.This article would further explore the factors which could induce the nuclear translocation of PAC1-R and the possible biological effects mediated by PAC1-R nuclear translocation.Methods:(1)Firstly,immunofluorescence was used to detect the induction of PAC1-R nuclear translocation by PACAP38 with different concentrations(10n M,100 n M,1?M)on PAC1-eGFPCHO specifically expressing PAC1-R,and Western blot and Multifunctional bioluminescence was used to detect the change of PAC1-R in each component of cells after PACAP38 treatment.And then it was detected in RGC-5 as well so as to compare whether the nuclear translocation was same as on PAC1-eGFP-CHO cells.(2)Secondly,PACAP(28-38),the C-terminal short peptide of PACAP38,and TAT(YGRKKRRQRRR)to induce the nuclear translocation of PAC1-R would be de detected on PAC1-eGFP-CHO cells.And they were compared with the effect of PO13,the N-terminal short peptide of PACAP38.(3)In addition,the induction of nuclear translocation of PAC1-R by small molecule positive allosteric regulator(sPAM1)synthesized by our research group was also measured on PAC1-eGFP-CHO cells and RGC-5 cells.(4)Immunofluorescence was then used to test the induction of nuclear translocation of PAC1-R on PAC1-eGFP-CHO cells by light,and some possible physiological response involved in nuclear translocation of PAC1-R were also explored.At the same time,western blot was used to detect the change of PAC1-R protein after blue light.(5)Verification of the overall level of PAC1-R during the induction of PAC1-R nuclear translocation by PACAP38 and sPAM1 was detected in RGC-5 cells.(6)On the basis of the experimental results and combining with previous studies,we would try to analyze the nuclear translocation mechanism of PAC1-R.Results:(1)It was found in PAC1-eGFP-CHO cells that gradient of concentrations of PACAP38(10 n M,100 n M,1 ?M)could induce PAC1-R to be effectively internalized and recruited in the periphery of the nuclear membrane,and then entered into nucleus.Meanwhile,in the process of nuclear translocation,it was concentration-dependent and dynamically cyclic.However,unlike PACAP38,which significantly induced the nuclear translocation of PAC1-R in PAC1-eGFPCHO cells,a statistically significant nuclear translocation effect could not be detected in RGC-5 cells.(2)TAT and PACAP(28-38)were both cationic arginine-rich peptides(CARPs),which could effectively induce the nuclear translocation of PAC1-R,while PO1,the short N-terminal peptide 3 of PACAP38,induced lower the rate of PAC1-R entering the nucleus than PACAP(28-38)and TAT.Corollary: The positive allosteric modulation(PAM)region of PAC1-R may be involved in mediating efficient nuclear translocation of PAC1-R itself.(3)Although sPAM1 was not CARPs and it was structurally bound to PAC1-R instead of the charge,sPAM1 could also induce nuclear translocation of PAC1-R.Not only could sPAM1 induce the nuclear translocation in PAC1-eGFP-CHO cells,but also in RGC-5 cells,initially confirming that the binding of PAM sites targeting the N-terminal extracellular domain of PAC1-R was involved in mediating effective nuclear translocation of PAC1-R.(4)Compared with red light,blue light could induce nuclear translocation of PAC1-R more effectively.After blue light induction,the position of PAC1-R in the nucleus overlapped with the position of chromosomal DNA;the PAC1-R in the nucleus was fragmented,and the degree of fragmentation at the C-terminus of the nucleus was proportionally related to the blue light exposure time.In addition,blue light-induced nuclear translocation of PAC1-R could be inhibited by ROS inhibitor NAC and palmitoylation inhibitor 2-BP.suggesting the nuclear translocation of PAC1-R induced by blue light may be related to reactive oxygen species and palmitoylation.(5)In RGC-5 cells,PACAP38 and sPAM1 could significantly induce the up-regulation of PAC1-R expression,suggesting that nuclear translocation of PAC1-R was involved in mediating up-regulation of PAC1-R expression.Conclusion: Factors such asPACAP38,TAT,PACAP(28-38),PO13,small molecule allosteric modulator(sPAM1)targeting PAC1-R and blue light could induce the nucleus translocation of PAC1-R,and it was concentration-dependent and dynamically cyclic.However,there was different induction efficiency of between TAT,PACAP(28-38)and PO13,suggesting that the PAM region of PAC1-R may be involved in mediating the efficient nuclear translocation of PAC1-R itself,which was initially confirmed in the process that sPAM1 significantly induced PAC1-R nuclear translocation.The nuclear translocation of PAC1-R may involve ROS and palmitoylation related to oxidative stress,and may lead to an increase in the activity of the PAC1-R promoter,which in turn leaded to up-regulate the PAC1-R expression and then to inspire subsequent signaling pathways and achieve response to stress.