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

PLoS One. 2014 Nov 26;9(11):e113913. doi: 10.1371/journal.pone.0113913. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Endocytosis
  • Mice
  • Mutation
  • Protein Multimerization
  • Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / genetics
  • Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / metabolism*
  • Wnt Proteins / metabolism*
  • Wnt Signaling Pathway*
  • beta Catenin / metabolism

Substances

  • Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I
  • Wnt Proteins
  • beta Catenin

Grants and funding

This work was supported by the National Natural Science Foundation of China (31100545 and 31200679), the Science and Technology Bureau of Guangdong Province (2011B090400024) and the Natural Science Foundation of Guangdong Province (S2011010002931). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.