Muscarinic acetylcholine 3 receptor (M3) displays agonist-dependent G protein signaling bias

Abstract ID 97436 Poster Board 157 Muscarinic acetylcholine 3 (M3) receptors on airway smooth muscle play a dominant role in mediating bronchoconstriction. M3 is known to couple to both Gai and Gaq/11 family members, but there is controversy over whether M3 also couples to Ga12/13. Bronchoconstriction is typically explained as a consequence of agonist binding to Gαq/11 coupled receptors and calcium-dependent phosphorylation of myosin light chain, however RhoA activation is also necessary for bronchoconstriction. Both Gaq/11 and Ga12/13 can activate the RhoA pathway, but Ga12/13 is the more potent activator. Characterization of G protein recruitment to M3 is relevant to airway physiology since inhibition of Rho kinase (ROCK), a critical downstream signaling event in the RhoA pathway, promotes bronchodilation while inhibition of either Gai or Gaq/11 signaling does not. Herein we report development of a split luciferase based M3 biosensor, which establishes definitively that both Ga₁₂ and Ga₁₃ couple to M3 in HEK293 Δ αq/11/12/13 cells. We also describe an improved, single-plasmid RhoA biosensor that enabled dissection of the contributions of Gaq and Ga12/13 to RhoA activation by carbachol, acetylcholine and methacholine in HEK293Δ αq/11/12/13 cells or in HEK293WT cells treated with the Gaq/11 specific depsipeptide inhibitor YM-254890. These analyses led to the surprising discovery that carbachol biases M3 towards recruitment of Gaq, while acetylcholine and methacholine bias M3 towards recruitment of Ga12/13. Although studies in animal models suggest that Ga₁₂ and Ga₁₃ expression increase as a function of asthma progression, we did not observe evidence of this in human airway smooth muscle cell lines derived from asthma patients. The data indicate that G protein selection by the M3 receptor is governed by agonist-modulated affinity and suggest that methacholine may be a more appropriate agonist for in vitro studies since its signaling better approximates that of the endogenous agonist acetylcholine. This study lays the foundation for functional characterization of Gαq/11 and Gα12/13 signaling in human airway smooth muscle, and for evaluating the role of G protein signaling bias in the efficacy of muscarinic agonists and antagonists.