Muscarinic Antagonist Mediated Activation of ERK Signaling Depends on β‐arrestin Recruitment to Augment Axonal Outgrowth in Neurons

Abstract

ObjectiveOne 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 (M1R) 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 M1R‐ERK signaling.MethodologyWe 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 GαS/L/Q/11/12/13 null HEK293 cell lines to dissect the downstream signaling events following antagonist binding to M1R.ResultsMT7 and pirenzepine‐mediated M1R‐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 M1R expressing β‐arrestin1/2 and GαS/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 M1R 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.ConclusionsOur study provides evidence that the neuritogenic effect of MT7 and pirenzepine may be mediated by selective blockade of M1R 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‐M1R‐ERK signaling could be exploited for improved therapy in neuropathic diseases.Support or Funding InformationSupported 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.