Muscarinic Antagonist Mediated Activation of ERK Signaling Depends on β‐arrestin Recruitment to Augment Axonal Outgrowth in Neurons
Bibliographic record
Abstract
Objective One of the major cellular effectors activated by G protein coupled receptors is extracellular signal‐regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and β‐arrestin mediated signaling pathways lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M 1 R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we hypothesized that the neuritogenic effect of muscarinic antagonists was mediated by M 1 R‐ERK signaling. Methodology We have used extensive two dimensional isoelectric focusing/SDS‐PAGE combined with analysis using multiple phospho‐epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector ‐ cyclic response element binding protein (CREB) in cultured adult rat primary dorsal root ganglion (DRG) neurons. Activation of CREB is known for neuroprotective and growth promoting effects. In addition, we have used CRISPR/Cas9 based β‐arrestin1/2 and Gα S/L/Q/11/12/13 null HEK293 cell lines to dissect the downstream signaling events following antagonist binding to M 1 R. Results MT7 and pirenzepine‐mediated M 1 R‐ERK signaling induced a significant increase in activation‐loop specific phosphorylation (T202/Y204) of ERK1/2 and significantly elevated pCREB(S133) levels in cultured sensory neurons. Further, using M 1 R expressing β‐arrestin1/2 and Gα S/L/Q/11/12/13 null HEK293 cell lines, we have shown that MT7 or pirenzepine mediated ERK1/2 activation is positively dependent on β‐arrestin recruitment to M 1 R but negatively regulated by G proteins. We have identified multiple charged fractions of ERK1/2 modulated by muscarinic antagonist treatment that is indicative of complex dynamics related to allosteric drug binding. Conclusions Our study provides evidence that the neuritogenic effect of MT7 and pirenzepine may be mediated by selective blockade of M 1 R and downstream activation of ERK‐CREB signaling. We also give insights into allosteric drug binding and complex dynamics of ERK activation. In addition, our study also indicates that removal of negative regulation by G proteins on antagonist‐M 1 R‐ERK signaling could be exploited for improved therapy in neuropathic diseases. Support or Funding Information Supported by CIHR grant # MOP‐130282 (PF) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".