Muscarinic acetylcholine receptor-mediated phosphorylation of extracellular signal-regulated kinase in HSY salivary ductal cells involves distinct signaling pathways

Rezon Yanuar; Shingo Semba; Akihiro Nezu; Akihiko Tanimura

doi:10.1016/j.job.2024.02.002

Muscarinic acetylcholine receptor-mediated phosphorylation of extracellular signal-regulated kinase in HSY salivary ductal cells involves distinct signaling pathways

J Oral Biosci. 2024 Feb 7:S1349-0079(24)00015-X. doi: 10.1016/j.job.2024.02.002. Online ahead of print.

Authors

Rezon Yanuar¹, Shingo Semba¹, Akihiro Nezu¹, Akihiko Tanimura²

Affiliations

¹ Division of Pharmacology, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan.
² Division of Pharmacology, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan. Electronic address: tanimura@hoku-iryo-u.ac.jp.

PMID: 38336259
DOI: 10.1016/j.job.2024.02.002

Abstract

Objectives: Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and β-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of β-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells.

Methods: HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42^MAPK and phosphor-p44/p42^MAPK.

Results: Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a βARK1/GRK2 inhibitor, barbadin (a β-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a β-arrestin inhibitor) and PP2 (a Src inhibitor).

Conclusion: Carbachol activates both G-protein and β-arrestin pathways, whereas pilocarpine exclusively activates the β-arrestin pathway. Additionally, downstream of β-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.

Keywords: Biased agonist; Extracellular signal-regulated kinase; Muscarinic acetylcholine receptor; Salivary ductal cells; β-arrestin.