PD 102807 Induces M3 mAChR-Dependent GRK-/Arrestin-Biased Signaling in Airway Smooth Muscle Cells

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 β-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 β-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 β (TGF-β)-mediated functions, PD 102807 inhibited TGF-β-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.

Keywords: COPD; acetylcholine; asthma; biased signaling.

Figure 1.

β-arrestin recruitment and calcium mobilization by muscarinic ligands. (A) Methacholine (MCh) (0.1, 1, 10, or 100 μM) and (B) PD 102807 (1, 10, or 100 μM) induce M3-mediated β-arrestin recruitment as assessed by bioluminescence resonance energy transfer (BRET) signal in HEK293 cells. (C) PD 102807 is a competitive antagonist of the M3 mAChR. Calcium mobilization in airway smooth muscle (ASM) cells by different concentrations of MCh in the presence or absence of PD 102807 (1, 10, or 100 μM; 15 min pretreatment) using Flexstation. (D) Maximal increase in β-arrestin recruitment BRET signal by mAChR ligands (100 μM) in HEK293 cells. (E) Net maximal calcium mobilization by mAChR ligands (100 μM) in human ASM cells stably expressing telomerase reverse transcriptase (hTERT) ASM cells. (F) Relationship between net maximal calcium mobilization and β-arrestin recruitment for each of the characterized mAChR ligands. Data are means ± SEM from two to four experiments. CCh = carbachol; MCh = methacholine; Musc = muscarine; OXO = oxotremorine sesquifumarate; OXO-M = oxotremorine-M; Pilo = pilocarpine; PD = PD 102807; Veh = vehicle.

Figure 2.

Methacholine and PD 102807 induce phosphorylated (p)-AMPK and p-ACC in a time- and concentration-dependent manner in human ASM cells; PD 102807–induced p-AMPK is M3 dependent. (A and B) Human hTERT ASM cell lines expressing M3 mAChRs were stimulated with (A) PD 102807 (100 μM) or (B) MCh (100 μM) for 0–240 min. (C–F) Cells were stimulated with increasing concentrations (1, 10, or 100 μM) of (D, F) MCh for 5 min or (C, E) PD 102807 for 20 min. *P < 0.05, **P < 0.01, and ***P < 0.001; one-way ANOVA followed by Dunnett’s multiple comparison test for differences from vehicle. (G) Cell lines expressing either high or low levels of M3 mAChR were stimulated with MCh (100 μM; 5 min) or PD 102807 (100 μM; 20 min). (H) In cell lines expressing low M3 mAChR, heterologous expression of M3 was achieved using lentiviral transfection. Transfected cells (control vector vs. M3 mAChR vector) were then stimulated with PD 102807 (100 μM; 20 min). (I) Cell lines expressing M3 mAChR were stimulated with PD 102807 (100 μM; 20 min) in the presence or absence of tiotropium bromide (100 nM; 30 min pretreatment). *P < 0.05, ***P < 0.001 versus vehicle, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001 versus own control; one-way ANOVA followed by Bonferroni multiple comparison test. Immunoblotting was performed using specific antibodies against p-AMPK, p-ERK, and p-ACC, as described in Methods. Representative blots are shown; loading was corrected for β-actin. Data are means ± SEM from three to seven experiments. Veh = vehicle.

Figure 3.

Gq inhibitor YM-254890 abrogates MCh-induced, but not PD 102807–induced, p-AMPK in human ASM cells expressing M3 mAChR. Human hTERT ASM cell lines were preincubated with YM-254890 (10 μM) for 15 min and then stimulated with (A) PD 102807 (100 μM; 20 min) or (B and C) MCh (100 μM; 5 min), and cell lysates were used for immunoblotting. Representative immunoblots for (A and B) p-AMPK and (C) p-ERK are shown; loading was corrected for β-actin. Data are means ± SEM from three to five experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus vehicle stimulation; ^##P < 0.01, and ^###P < 0.001 versus vehicle-pretreated, MCh-stimulated condition; one-way ANOVA followed by Bonferroni multiple comparison test. Veh = vehicle.

Figure 4.

Role of β-arrestin and GRK2/3 in PD 102807–induced or MCh-induced p-AMPK and p-ACC. After mock, β-arrestin-1, β-arrestin-2, or GRK2/3 siRNA transfection, cells were stimulated with (A–C) PD 102807 (100 μM; 20 min), or (D–F) MCh (100 μM; 5 min), and (A, B, D, and E) p-AMPK and (C and F) p-ACC were assessed by immunoblotting. (G) β-arrestin-1, β-arrestin-2, and GRK2/3 knockdowns were confirmed by immunoblotting. Representative immunoblots for p-AMPK and p-ACC are shown; loading was corrected for total AMPK or β-actin. Data are means ± SEM from three to six experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus vehicle stimulation; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 versus mock-transfected PD 102807–stimulated condition; one-way ANOVA followed by Bonferroni multiple comparison test. Veh = vehicle.

Figure 5.

PD 102807 inhibits TGF-β–induced signaling and function in ASM cells in an M3-dependent manner. (A) SMAD-Luc transfected human hTERT ASM cell lines were preincubated with PD 102807 (1 or 10 μM) for 20 min and then stimulated with TGF-β (1 ng/mL) for 6 hours. Luciferase activity was determined as described in Methods. (B) hTERT ASM cell lines were treated with TGF-β (1 ng/mL) ± PD 102807 (10 μM) for 3 days. Cells were then fixed and permeabilized, and smooth muscle α-actin (α-SMA) (488 nm; green) and phalloidin (594 nm; red) expression was determined by immunofluorescence. Cells were counterstained with DAPI (blue) as described in Methods. Representative images are shown. (C) hTERT ASM cells were stimulated with TGF-β (1 ng/mL) with or without MCh (10 μM) or PD 102807 (10 μM) for 3 days, and α-SMA expression was determined by immunoblotting. Representative immunoblots for α-SMA are shown; loading was corrected for β-actin. (D) ASM cell contraction was after cells were treated with TGF-β (1 ng/mL) with or without PD 102807 (10 μM) for 3 days. Collagen gel contraction data are shown as percent reduction in gel area after histamine stimulation (1 μM; 10 min). Data are means ± SEM from four to five experiments. *P < 0.05 and ****P < 0.0001 versus vehicle-stimulated condition; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 versus vehicle-pretreated TGF-β condition; one-way ANOVA followed by Bonferroni multiple comparison test. Veh = vehicle.

Figure 6.

GRK2/3 knockdown attenuates PD 102807–mediated inhibition of TGF-β–induced α-SMA expression. Following (A) GRK2/3 siRNA transfection, ASM cells were stimulated with TGF-β (1 ng/ml) for 3 days in the presence or absence of PD 102807 (10 μM), and α-SMA expression was determined by immunoblotting. (B) GRK2/3 knockdown after the 3-day treatment (Day 6 post–siRNA transfection) was confirmed by immunoblotting. Representative immunoblots for α-SMA are shown; loading was corrected for β-actin. Data are means ± SEM from four experiments. *P < 0.05; ***P < 0.001 versus vehicle pretreated TGF-β condition; ^#P < 0.05 versus mock-transfected, PD 102807–pretreated TGF-β–stimulated condition; one-way ANOVA followed by Bonferroni multiple comparison test. Veh = vehicle.

Figure 7.

PD 102807 is an M3 mAChR biased ligand that inhibits M3-mediated Gq signaling and promotes (Gq-independent) GRK2/3-arrestin–dependent signaling. M3 mAChR agonists, such as MCh, promote Gq-mediated calcium mobilization and ASM contraction (left side). In addition, MCh induces a transient phosphorylation of AMPK that is Gq calcium dependent. The biased M3 ligand PD 102807, on the other hand, acts as an orthosteric antagonist for M3-Gq–dependent signaling and promotes GRK2/3-arrestin–dependent sustained AMPK signaling (right side). TGF-β–induced signaling, mediated by TGF-β receptors II and I (TβRII/I), promotes phenotype switching to a hypercontractile ASM phenotype. PD 102807–induced GRK2/3-arrestin–dependent AMPK signaling inhibits TGF-β signaling and thereby attenuates TGF-β–induced ASM hypercontractility.