ABSTRACT
Airway smooth muscle (ASM) cells attain a hypercontractile phenotype during obstructive airway diseases. We recently identified a biased M3 muscarinic acetylcholine receptor (mAChR) ligand, PD 102807, that induces GRK-/arrestin-dependent AMP-activated protein kinase (AMPK) activation to inhibit transforming growth factor-ß-induced hypercontractile ASM phenotype. Conversely, the balanced mAChR agonist, methacholine (MCh), activates AMPK yet does not regulate ASM phenotype. In the current study, we demonstrate that PD 102807- and MCh-induced AMPK activation both depend on Ca2+/calmodulin-dependent kinase kinases (CaMKKs). However, MCh-induced AMPK activation is calcium-dependent and mediated by CaMKK1 and CaMKK2 isoforms. In contrast, PD 102807-induced signaling is calcium-independent and mediated by the atypical subtype protein kinase C-iota and the CaMKK1 (but not CaMKK2) isoform. Both MCh- and PD 102807-induced AMPK activation involve the AMPK α1 isoform. PD 102807-induced AMPK α1 (but not AMPK α2) isoform activation mediates inhibition of the mammalian target of rapamycin complex 1 (mTORC1) in ASM cells, as demonstrated by increased Raptor (regulatory-associated protein of mTOR) phosphorylation as well as inhibition of phospho-S6 protein and serum response element-luciferase activity. The mTORC1 inhibitor rapamycin and the AMPK activator metformin both mimic the ability of PD 102807 to attenuate transforming growth factor-ß-induced α-smooth muscle actin expression (a marker of hypercontractile ASM). These data indicate that PD 102807 transduces a signaling pathway (AMPK-mediated mTORC1 inhibition) qualitatively distinct from canonical M3 mAChR signaling to prevent pathogenic remodeling of ASM, thus demonstrating PD 102807 is a biased M3 mAChR ligand with therapeutic potential for the management of obstructive airway disease.
ABSTRACT
G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated ß-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by ß-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor ß (TGF-ß)-mediated functions, PD 102807 inhibited TGF-ß-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.
