Antimuscarinic drugs exert β-arrestin-biased agonism at the muscarinic acetylcholine type 1 receptor

Previous studies indicate that both pirenzepine (PZ), a selective orthosteric muscarinic acetylcholine type 1 receptor (M1R) antagonist, and muscarinic toxin 7 (MT7), a negative M1R allosteric modulator (NAM), act via M1R to promote neuritogenesis in cultured adult rodent primary dorsal root ganglia (DRG) sensory neurons, in part, through {beta}-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 {beta}-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 {beta}-arrestin2 to M1R (analyzed using nano-BRET) and increased ERK phosphorylation in both HEK293 cells and DRG neurons. DRG neurons of different sub-types express M1R, and ERK activation by MT7 was only observed in M1R-positive neurons. These novel pharmacological effects occurred in the absence of activation of G protein signaling or receptor internalization. PZ phosphorylated M1R at six specific serine/threonine residues (T230, S251, T254, S321, T354, S356) of intracellular loop 3 (ICL3) and deletion mutation of these sites suppressed PZ and MT7 induction of {beta}-arrestin binding to M1R and inhibited ERK activation. With regard to PZ signaling, alanine substitution at S251 and T254 was sufficient to impede {beta}-arrestin binding and ERK activation. {beta}-arrestin-biased activity of PZ and MT7 involved the mobilization of casein kinase 2 (CK2) and this occurred in the absence of Gq or G protein receptor kinase (GRK) activity. Pharmacological or siRNA-based inhibition of CK2 blocked PZ-induction of {beta}-arrestin association, ERK activation and neurite outgrowth in DRG neurons. In conclusion, PZ/MT7 activated M1R toward the {beta}-arrestin signaling pathway in both HEK293 cells and DRG neurons to augment ERK activation and neurite outgrowth via engagement of CK2. One-sentence summaryAntimuscarinic drugs act as {beta}-arrestin-biased agonists via casein kinase 2 activation to promote ERK1/2 phosphorylation and neurite outgrowth O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/649213v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1bb8da7org.highwire.dtl.DTLVardef@510218org.highwire.dtl.DTLVardef@608d69org.highwire.dtl.DTLVardef@e40d9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Schematic presentation of the effect of muscarinic ligands at M1R associated signaling pathway. (A) Muscarine/carbachol acts as a balanced ligand by engaging both Gq and -arrestin signaling pathways and treatment with pirenzepine/MT7 blocks these effects. (B) Pirenzepine/MT7 acts as a -arrestin biased ligand by 1) phosphorylating of ICL3 region of M1R via CK2 (but not GRKs), 2) no activation of G protein signaling, 3) recruitment of -arrestin 2 and 4) ERK1/2 activation leading to neurite outgrowth in DRG sensory neurons. This figure was generated by BioRender under license number EK285MUOPQ. C_FIG