The Mechanism Of Intrinsic/Basal Activity Of B Class GPCR PAC1 And Its Regulation By Doxycycline

Objective: B class G-protein-coupled receptor(GPCR) PAC1 is PACAP(pituitary adenylate cyclase-activating polypeptide)-preferring receptor. Its high expression is always associated with nerve injury and tumors. Our previous research confirmed the dimerization of PAC1 and found that the M-PAC1 mutation in the N-terminal first Cys/Ala lost the ability to form dimers. We have confirmed that the dimerization of PAC1 has intrinsic/basal activity, but we have no idea about the mechanism of the intrinsic activity. Occasionally, It was found that 10-1000ng/ml doxycycline(Dox) improve the anti-apoptotic ability in the cells expressing PAC1, which was related with the dimer-dependent intrinsic/basal activity of PAC1. In this research, therefore, we clarified the mechanism of dimer-dependent intrinsic/basal activity of PAC1 and the effects of Dox on PAC1’s intrinsic/basal activity. Method: In the apoptosis induced.by serum withdrawal in the PAC1-CHO, M-PAC1-CHO and pc DNA-CHO cells, series of inhibitors of transduction signal pathways and PAC1 dimerization inhibitor acetylcysteine(NAC) combined with the assays on the apoptotic indexs including the cells viability remained determined by MTT, Caspase 3 activity and Bcl-2 level were used to screen the signal pathway involved in PAC1-dimer-dependent intrinsic activity. Signal pathway inhibitors combined with Western bolt of the key proteins of related signal pathway and TOP-flash assay forβ-catenin signals and down-regulation of PAC1 express using sh RNA in Neuro2 a were also used to detect the molecular mechanmis of PAC1 ’s intrinsic activity, following which, the effects of Dox on the intrinsic activity of PAC1 was analyzed Result: Only PAC1-CHO cells expressing PAC1 dimers showed anti-apoptotic activity against serum withdawl induced apoptosis, while M-PAC1-CHO cells and CHO cells lack in PAC1 dimers did not. Wnt/ β-catenin pathway inhibitor XAV939 and NAC significantly inhibited the antiapoptotic ability of PAC1-CHO cells. The results of Western blot and TOP-flash showed that XAV939 and NAC significantly decreased the signals of Wnt/ β-catenin pathway The antiapoptotic ability and the signalf Wnt/ β-catenin pathway were lowered significantly by sh RNA of PAC1 in Neuro2 a cells. In research on Dox with PAC1, it was found that 10-1000ng/ml Dox up-regulated the expression of PAC1 and induced the endocytosis of PAC1. And the higher concentration of Dox resulted into stronger antiapoptotic ability and stronger signal of Wnt/ β-catenin pathway in PAC1-CHO cells. The declined expression of PAC1 by sh RNA of PAC1 inhibited the enhanced effects of against antiapoptosis in Neuro2 a. Conclusion: PAC1 had ligand-independent, dimer-dependent intrinsic activity, which was related to Wnt/ β-catenin pathway. It was found for the first time that Dox promoted the expression of PAC1 and exerted the anti-apoptotic activity through PAC1. The investigation on the mechanism of the intrinsic activity of PAC1 and the finding of the effects of Dox on the expression and the activity of PAC1 help the further drug development targeting PAC1.