PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization

Atsuko Hayata-Takano,Keita Moriguchi,Naoki Encho,Takanobu Nakazawa,Kohei Kitagawa,Yusuke Shintani,Hitoshi Hashimoto

doi:10.3389/fendo.2021.732456

Abstract

Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) display psychomotor abnormalities, most of which are ameliorated by atypical antipsychotics with serotonin (5-HT) 2A receptor (5-HT2A) antagonism. Heterozygous Pacap mutant mice show a significantly higher hallucinogenic response than wild-type mice to a 5-HT2A agonist. Endogenous PACAP may, therefore, affect 5-HT2A signaling; however, the underlying neurobiological mechanism for this remains unclear. Here, we examined whether PACAP modulates 5-HT2A signaling by addressing cellular protein localization. PACAP induced an increase in internalization of 5-HT2A but not 5-HT1A, 5-HT2C, dopamine D2 receptors or metabotropic glutamate receptor 2 in HEK293T cells. This PACAP action was inhibited by protein kinase C inhibitors, β-arrestin2 silencing, the PACAP receptor PAC1 antagonist PACAP6-38, and PAC1 silencing. In addition, the levels of endogenous 5-HT2A were decreased on the cell surface of primary cultured cortical neurons after PACAP stimulation and were increased in frontal cortex cell membranes of Pacap−/− mice. Finally, intracerebroventricular PACAP administration suppressed 5-HT2A agonist-induced head twitch responses in mice. These results suggest that PACAP–PAC1 signaling increases 5-HT2A internalization resulting in attenuation of 5-HT2A-mediated signaling, although further study is necessary to determine the relationship between behavioral abnormalities in Pacap−/− mice and PACAP-induced 5-HT2A internalization.

Highlights

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that regulates a wide array of physiological responses, including emotion, cognition and motor function
We examined whether PAC1, VPAC1, VPAC2, and 5-HT 2A receptor (5-HT2A) mRNAs were expressed in HEK293T cells using reverse transcription (RT)-PCR analysis
Quantitative RT-PCR analysis showed that PC12 cells and SH-SY5Y cells expressed relatively higher levels of PAC1 mRNA as expected from the previous reports [27,28,29], and both our HEK293T cell cultures and the HEK293T cells provided by RIKEN BRC Cell Bank (RCB2202; the National Bio-Resource Project of the MEXT/AMED, Japan) moderately expressed PAC1 mRNA at similar levels

Summary

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that regulates a wide array of physiological responses, including emotion, cognition and motor function. Pacap−/− mice have increased 5-HT content and 5-HT-immunoreactive cell counts in the dorsal raphe [12] and slightly decreased levels of the 5HT metabolite, 5-hydroxyindoleacetic acid, in the cortex and striatum [5]. These findings indicate that 5-HT2A function may be involved in psychiatric conditions in which PACAP signaling is dysfunctional and that functional crosstalk may exist between PACAP and 5-HT2A signaling pathways.

Methods

Results

Conclusion