Antimuscarinic drugs exert β-arrestin–biased agonism at the muscarinic acetylcholine type 1 receptor to promote DRG neuritogenesis

Pirenzepine (PZ) and muscarinic toxin 7 (MT7) are muscarinic acetylcholine type 1 receptor (M₁R) antagonists that promote neuritogenesis in primary adult rodent dorsal root ganglion (DRG) sensory neurons, in part through β-arrestin-dependent activation of ERK1/2. Here, we found that PZ and MT7 exhibited β-arrestin-biased agonism at M₁R. PZ and MT7 recruited β-arrestin 2 to M₁R and increased ERK phosphorylation in both HEK293 cells and DRG neurons in a concentration-dependent manner. Moreover, ERK activation by MT7 occurred only in M₁R-positive DRG neurons and did not require G protein signaling or receptor internalization. PZ stimulated M₁R phosphorylation at multiple serine and threonine residues. Mutation of Ser²⁵¹ and Thr²⁵⁴ in M₁R suppressed PZ- and MT7-dependent activation of β-arrestins and PZ-dependent β-arrestin binding and ERK activation. The β-arrestin-biased activities of PZ and MT7 required the activity of casein kinase 2 (CK2) but not that of Gα_q or GPCR kinases (GRKs). Pharmacological or siRNA-based inhibition of CK2 blocked PZ-dependent β-arrestin recruitment, ERK activation, and neurite outgrowth in DRG neurons. These results implicate a GRK- and G protein-independent mechanism for the β-arrestin-biased agonism and antimuscarinic effects of PZ and MT7.