title: Dimer-dependent intrinsic/basal activity of the class B G protein-coupled receptor PAC1 promotes cellular anti-apoptotic activity through Wnt/β-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 β-catenin, cyclin D1 and c-myc levels corresponding to the Wnt/β-catenin signal than did M-PAC1-CHO. In addition, the Wnt/β-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/β-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/β-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/β-catenin signal. A similar correlation was also found with the down-regulation of PAC1 in the Neuro2a neuroblastoma cell. BiFC combined with fluorescence confocal imaging indicated that during serum-withdrawal-induced apoptosis, PAC1 dimers displayed significant endocytosis. These findings indicate that PAC1 has ligand-independent and dimer-dependent intrinsic/basal activity, conferring cells with anti-apoptotic activities against serum withdrawal, which is involved in the Wnt/β-catenin signal and is associated with the endocytosis of PAC1 dimers. The discovery and study of the dimer-dependent basal activity of PAC1 not only help us understand the physiological and pathological role of PAC1 but also promote the development of drugs targeting PAC1.
authors.affiliation: Institute of Biomedicine, Department of Cell Biology, Jinan University, Guangzhou, China.
authors.forename: Rongjie
authors.initials: R
authors.lastname: Yu
authors.affiliation: Institute of Biomedicine, Department of Cell Biology, Jinan University, Guangzhou, China.
authors.forename: Zekai
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Institute of Biomedicine, Department of Cell Biology, Jinan University, Guangzhou, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.affiliation: Institute of Biomedicine, Department of Cell Biology, Jinan University, Guangzhou, China.
authors.forename: Yanxu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Biomedicine, Department of Cell Biology, Jinan University, Guangzhou, China.
authors.forename: Jiaping
authors.initials: J
authors.lastname: Zhong
mesh_terms: D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D051379:Mice; D009154:Mutation; D055503:Protein Multimerization; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000235:genetics / Q000378:metabolism*; D051153:Wnt Proteins / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D051153:Wnt Proteins; D051176:beta Catenin