Antimuscarinic drugs exert β-arrestin-biased agonism at the muscarinic acetylcholine type 1 receptor
Bibliographic record
Abstract
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 Galphaq 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".