Cell-penetrating Peptide TAT Exerts Neuroprotective Effect Through Its Allosteric Modulation On Neuropeptide GPCR PAC1-R

Objective:Pituitary adenylate cyclase activating polypeptide?PACAP?receptor 1?PAC1-R?belonging to class B G protein-coupled receptors?GPCRs?is PACAP preferring receptor and mediates the significant neuroprotective activity,which is considered as the drug target of nervous system diseases.When used cell penetrating peptide TAT?YGRKKRRQRRR?to enhance the traversing activity of neuropeptide vasoactive intestinal peptide?VIP?,found by chance that TAT tagging?VIP-TAT?facilitated the activation of PAC1-R and TAT had similar two-dimension structure with PACAP?28-38?.So in this research,used computer molecular docking,microscale thermophoresis?MST?assay and cAMP assay to identify the hypothesis that TAT worked as an allosteric modulator of PAC1-R.Method:PAC1-R shRNA and PAC1-R antagonist PACAP?6-38?were used to determine whether PAC1-R mediated the neuroprotective activities of TAT and VIP-TAT in mouse neuroblastoma Neuro2a cells against scopolamine-induced apoptosis.The Computer molecular docking and MST assay were used to determine the binding of TAT with the N-terminal extracellular domain of PAC1-R?PAC1-EC1?.Then Chinese hamster ovary cells PAC1-CHO with high expression of PAC1-R and enhanced green fluorescent protein?eGFP?fusion protein,combined with cAMP assay were used to detect whether TAT alone and its cooperation with VIP may work as a positive allosteric modulator?PAM?on the activation of PAC1-R.And a series of endocytosis inhibitors,including clathrin inhibitor chlorpromazine,caveolae inhibitor?-cyclodextrin and macropinocytosis inhibitor wortmannin were used for the further exploration of the internalization mechanism of PAC1-R induced by TAT and VIP-TAT.Result:Both PAC1-R shRNA and PAC1-R antagonist PACAP?6-38?significantly inhibited the neuroprotective activity of TAT?1nM?and VIP-TAT?1nM?against the scopolamine-induced Neuro2a apoptosis,confirming that PAC1-R mediates the neuroprotective activities of TAT and VIP-TAT.The computer docking indicated that the binding site of TAT on PAC1-EC1 is similar to that of PACAP?28-38?.The MST assay results showed that the affinity of TAT-binding PAC1-ECI with KD values of 127.0+/-12.3?M was similar to that of PACAP?28-38?with KD of107.0+/-13.4?M.The experiments on allosteric modulation effects of TAT on PAC1-R showed that TAT alone induced cAMP accumulation in PAC1-CHO cells with an EC₅₀ of 0.063±0.012nM and the maximum effect lower than 50%of the maximum effect of PACAP27.Furthermore,the synergic effect EC₂₀VIP+TAT was not significantly stronger than that of TAT alone,while VIP-TAT increased intracellular cAMP levels with an EC₅₀ of 52.6±8.6 pM,which is about1/500 of that of VIP(EC₅₀ VIP=30.1±9.1 nM).These results indicated that the binding of TAT with PAC1-R was prior to the binding of VIP with PAC1-R,and the binding of TAT induced the rapid internalization of PAC1-R,which inhibited the further binding of PAC1-R with VIP.Thus TAT alone worked as a PAC1-R allosteric agonist and its non-covalent linkage with VIP did not have a positive allosteric effect on the activation of PAC1-R.Only the covalent tagging of TAT to VIP had a positive allosteric regulatory effect on PAC1-R.Further research on the internalization of PAC1-R revealed that TAT in low concentration?0.01nM-0.1nM?induced PAC1-R internalization more effectively than VIP and PACAP?28-38?.And the clathrin inhibitor chlorpromazine significantly inhibited the internalization of PAC1-R and cAMP accumulation induced by VIP or VIP-TAT,but did not inhibit the internalization of PAC1-R and cAMP accumulation induced by TAT;while only the caveolae inhibitor?-cyclodextrin significantly inhibited TAT?1nM?-induced internalization of PAC1-R.All these results indicated that TAT alone and TAT covalent linkage with VIP mediated different internalization and signal transduction pathways of PAC1-R from each other.Conclusion:PAC1-R was confirmed to be involved in the neuroprotective activities of TAT and VIP-TAT.The binding of TAT to PAC1-EC1 was confirmed for the first time.TAT alone played the role as an allosteric agonists of PAC1-R through caveolae-mediated internalization and signal transduction pathway;while covalent tagging TAT to VIP exerts its effect as the PAM of PAC1-R through clathrin-mediated internalization and signal transduction pathways.The determination of the allosteric modulation effects of TAT on PAC1-R not only helps to explain the neuroprotective activity of TAT,but also contributes to the use of TAT in the development of drugs targeting the PAC1–R for nervous system disorders or diseases.