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

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. 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. 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). 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. [Display omitted] • Muscarinic receptors agonists carbachol and pilocarpine induce phosphorylation of ERK1/2. • Carbachol activates two pathways, the G protein- and β-arrestin-mediated pathways. • Pilocarpine preferentially activates β-arrestin-mediated pathways. • Carbachol and pilocarpine activate different pathways downstream of β-arrestin in HSY cells. [ABSTRACT FROM AUTHOR]