Amiri, Shayan2025-09-042025-09-042025-08-212025-08-21http://hdl.handle.net/1993/39296The M1 muscarinic acetylcholine receptor (M1R) is highly expressed in the nervous system, and has been studied to discover new therapeutic drugs for a range of neurological and psychiatric disorders. Studies from our lab and others also highlighted the fact that antagonists of M1R could be therapeutic agents for neuropathies. However, the mechanism(s) of action through which M1R antagonists exert their therapeutic effects are not well understood. Previous studies indicate that both pirenzepine (PZ), a selective M1R antagonist, and muscarinic toxin 7 (MT7), a negative M1R allosteric modulator, act via M1R to promote neuritogenesis in rodent primary dorsal root ganglia (DRG) sensory neurons, in part, through β-arrestin-dependent activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Furthermore, these antagonists reverse nerve degeneration in a variety of rodent models of peripheral neuropathy through multiple complementary pathways. To understand the therapeutic effects and mechanism of M1R antagonist-induced ERK1/2 phosphorylation, we tested the hypothesis that PZ and MT7 possess β-arrestin-biased agonism at M1R to drive activation of ERK and enhance neurite outgrowth. Treatment for up to 30 min with PZ and MT7 dose-dependently recruited β-arrestin2 to M1R (analyzed using nano-BRET) and increased ERK phosphorylation in both HEK293 cells and DRG neurons. These novel pharmacological effects occurred in the absence of activation of G protein signaling or receptor internalization. PZ promoted M1R phosphorylation at six specific serine/threonine residues (Thr230, Ser251, Thr254, Ser321, Thr354, Ser356) and deletion mutation of these sites suppressed PZ and MT7 induction of β-arrestin binding to M1R and inhibited ERK activation. β-arrestin-biased activity of PZ and MT7 involved the mobilization of casein kinase 2 (CK2) and this occurred in the absence of Gαq or G protein receptor kinase (GRK) activity. Pharmacological or siRNA-based inhibition of CK2 blocked PZ-induction of β-arrestin association, ERK activation and neurite outgrowth in DRG neurons. In conclusion, PZ/MT7 activated M1R toward the β-arrestin signaling pathway in both HEK293 cells and DRG neurons to augment ERK activation and neurite outgrowth via engagement of CK2. Results of this work provide novel findings with far reaching consequences on our understanding of the signaling mechanism of antimuscarinic drugs.engM1 muscarinic acetylcholine receptorBiased agonismβ-arrestinDorsal root ganglia sensory neurons (DRG)Muscarinic antagonists