Dimer-dependent intrinsic/basal activity of the class b g protein-coupled receptor pac1 promotes cellular anti-apoptotic activity through wnt/b-catenin pathways that are associated with dimer endocytosis

Abstract

The high expression of PACAP (pituitary adenylate cyclase-activating polypeptide)- preferring receptor PAC1 is associated with nerve injury and tumors. Our previous report (Yu R, et al. PLoS One 2012; 7: e51811) 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. In this study, Chinese hamster ovary (CHO-K1) cells overexpressing wild-type PAC1 (PAC1-CHO) had significantly higher anti-apoptotic activities against serum withdrawal-induced apoptosis associated with a lower caspase 3 activity and a higher Bcl-2 level in a ligand-independent manner than those of CHO cells overexpressing the mutant M-PAC1 (M-PAC1-CHO). PAC1-CHO had significantly higher b-catenin, cyclin D1 and c-myc levels corresponding to the Wnt/b-catenin signal than did M-PAC1-CHO. In addition, the Wnt/b-catenin pathway inhibitor XAV939 significantly inhibited the anti-apoptotic activities of PAC1-CHO. Top-flash assays demonstrated that PAC1-CHO had a significantly stronger Wnt/b-catenin signal than did M-PAC1-CHO. Acetylcysteine (NAC) as an inhibitor of the dimerization of PAC1 inhibited the anti-apoptotic activities that were endowed by PAC1 and decreased the Wnt/b-catenin signal in Top-flash assays. In the PAC1 Tet (tetracycline)-on inducible gene expression system by doxycycline (Dox), higher expression levels of PAC1 resulted in higher anti-apoptotic activities that were associated with a stronger Wnt/b-catenin signal. A similar correlation was also found with the down-regulation of PAC1 in the Neuro2a neuroblastoma cell. @copy;2014 Yu et al.