Conformational Rearrangements and Signaling Cascades Involved in Ligand-Biased Mitogen-Activated Protein Kinase Signaling through the 1-Adrenergic Receptor

In recent years, several studies have demonstrated that different ligands can have distinct efficacy profiles toward various signaling pathways through a unique receptor. For example, 1-adrenergic compounds that are inverse agonists toward the adenylyl cyclase (AC) can display agonist activity for the mitogen-activated protein kinase (MAPK) pathway. Such a phenomenon, often termed functional selectivity, has now been clearly established for many G protein-coupled receptors when considering distinct signaling output. However, the possibility that ligands could selectively engage distinct effectors to activate a single signaling output by promoting specific receptor conformations has not been extensively examined. Here, we took advantage of the fact that isoproterenol, bucindolol and propranolol (full, partial, and inverse agonists for the AC pathway, respectively) all activate MAPK through the 1-adrenergic receptor ( 1AR) to probe such conformational-biased signaling. Although the three compounds stimulated MAPK in a srcdependent manner, isoproterenol acted through both G i and G protein-independent pathways, whereas bucindolol and propranolol promoted MAPK activation through the G proteinindependent pathway only. The existence of such distinct signaling cascades linking 1AR to MAPK activation was correlated with ligand-specific conformational rearrangements of receptor/G protein complexes measured by bioluminescence resonance energy transfer. Taken together, our data indicate that discrete local conformational changes can selectively promote the recruitment of distinct proximal signaling partners that can engage distinct signaling outputs and/or converge on the same signaling output. G protein-coupled receptors (GPCRs) represent the most prevalent class of transmembrane signaling proteins. They can modulate a large variety of signaling systems to ensure a fine regulation of cell function in response to external stimuli. This signaling diversity is achieved in part by the capacity of one receptor to couple to diverse G proteins and non-G protein effectors (Bockaert et al., 2004). Over the past decade, many studies have demonstrated that different subsets of these effector systems can be selectively modulated by distinct ligands through a unique receptor, a phenomenon often referred to as ligand-biased signaling (Galandrin et al., 2007; Kenakin, 2007). Hence, efficacy of GPCR ligands is increasingly considered as a pluridimensional parameter that should include in its definition, in addition to the ligand/ receptor pair, the signaling pathways considered. Characterizing a panel of -adrenergic ligands for their efficacy profiles toward two of the 1-adrenergic receptor ( 1AR)-stimulated signaling pathways, adenylyl cyclase (AC) and mitogen-activated protein kinase (MAPK) pathThis work was supported by grants from the Canadian Institute for Health Research (CIHR) and the Québec Heart and Stroke Foundation. G.O.-L. was supported by a studentship from the CIHR; K.O. was an invited researcher from Tanabe Seiyaku Co. Ltd.; and M.B. holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology. C.G. and M.B. contributed equally to this work. 1 Current affiliation: Institut National de la Santé et de la Recherche Médicale U858-I2MR-Equipe 8, Toulouse, France. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.043893. ABBREVIATIONS: GPCR, G protein-coupled receptor; AR, -adrenergic receptor; AC, adenylyl cyclase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; BRET, bioluminescence resonance energy transfer; AVP, arginine 8 vasopressin; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PD98059, 2 -amino-3 -methoxyflavone; EGF, epidermal growth factor; CTX, cholera toxin B; PTX, pertussis toxin; PKA, protein kinase A; HRP, horseradish peroxidase; GFP, green fluorescent protein; GFP10, blue-shifted mutant of green fluorescent protein; ARK, -adrenergic receptor kinase; HEK, human embryonic kidney; OR, opioid receptor; V2R, vasopressin type 2 receptor; PTH, parathyroid hormone; siRNA, small interfering RNA; PBS, phosphate-buffered saline; Rluc, Renilla reniformis luciferase; TBS-T, Tris-buffered saline/Tween 20; p-, phospho; MEK, mitogen-activated protein kinase kinase. 0026-895X/08/7401-162–172$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 43893/3353587 Mol Pharmacol 74:162–172, 2008 Printed in U.S.A. 162 at A PE T Jornals on A uust 6, 2017 m oharm .aspeurnals.org D ow nladed from ways, we found that compounds displaying opposite efficacy toward AC shared common agonistic activity for MAPK. Indeed, isoproterenol, bucindolol, and propranolol that are, respectively, full agonist, partial agonist, and inverse agonists for AC were found to act as agonists for the extracellular signal-regulated kinase (ERK) 1/2 pathway (Galandrin and Bouvier, 2006). The inverse efficacy of propranolol compared with bucindolol and isoproterenol for 1AR-stimulated AC clearly reveals the ability of the compounds to promote distinct receptor conformations. Because the three compounds activate MAPK, the data indicate that distinct receptor conformations can converge on the stimulation of a single downstream effector system. Multiple pathways have been shown to link GPCR to MAPK activation. Some of these involve the generation of second messengers resulting from classic G protein activation, whereas others rely on the scaffolding properties of proteins such as arrestin (Luttrell, 2005). However, the correlation between the signaling cascades leading to MAPK and specific receptor conformation that can be promoted by different ligands has not been established yet. We therefore took advantage of the fact that isoproterenol-, bucindolol-, and propranolol-promoted receptor conformations converged on ERK1/2 stimulation to link receptor conformations to specific effector cascades. For this purpose, the pathways leading to the 1AR-mediated activation of ERK1/2 were investigated for the three ligands, whereas the conformations of the liganded receptor were assessed by bioluminescence resonance energy transfer (BRET) measurements monitoring structural rearrangements within receptor/G protein complexes. We report that isoproterenol stimulated ERK1/2 through both Gi-dependent and G protein-independent mechanisms, whereas bucindolol and propranolol engaged MAPK only via the G protein-independent pathway. Note that the distinct signaling pattern of isoproterenol was associated with a unique conformational signature of the receptor/G protein complex, confirming that distinct ligands can select different signaling cascades by promoting discrete conformational rearrangements. Materials and Methods Reagents. ( )-Isoproterenol, DL-propranolol, Leu-enkephalin, and arginine-8 vasopressin (AVP) were purchased from Sigma-Aldrich (St. Louis, MO), whereas bucindolol was a generous gift from Dr. Michael Bristow (University of Colorado Health Sciences Center, Aurora, CO). Recombinant human epidermal growth factor (EGF) was from PeproTech (Rocky Hill, NJ). Cholera toxin B subunit (CTX) and pertussis toxin (PTX) were from Sigma-Aldrich. Inhibitors PP2 and PD98059 were from Calbiochem (San Diego, CA). Mouse antiphosphorylated ERK1/2, rabbit anti-ERK1/2, and antiarrestin2 (H9) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody recognizing the phosphor-(Ser/Thr) protein kinase A (PKA) substrate was purchased from Cell Signaling Technology Inc. (Danvers, MA). HRP-anti-mouse and HRP-anti-rabbit polyclonal antibodies were from GE Healthcare (Baie d’Urfé, QC, Canada). All other reagents were of analytical grade, and they were obtained from various suppliers. Expression Vectors. The plasmid encoding rat ERK2-GFP (DeFea et al., 2000b) and the -adrenergic receptor kinase ( ARK) carboxyl-terminal (C)-tail conjugated to the extracellular and transmembrane domain of the CD8 protein (T8 ARKctail) (Crespo et al., 1995) were generous gifts from K. DeFea (University of California, Riverside, CA) and J. S. Gutkind (National Institutes of Health, Bethesda, MD), respectively. Plasmid encoding arrestin1 deleted in its C-tail from amino acid 319 to amino acid 418 ( -Arr 318-419) was generously provided by Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia, PA). Plasmids encoding the 1-adrenergic receptor fused to its C terminus with humanized Renilla reniformis luciferase ( 1AR-hRLuc) or the blue variant of GFP ( 1AR-GFP10) were described previously (Mercier et al., 2002). The expression vectors containing human G protein subunits (G i1, G 2, and G 1) were obtained from Missouri University of Science and Technology (Rolla, MO). Plasmids encoding G 2 fused at its N terminus to GFP10 (GFP10-G 2) or G i1 tagged with hRLuc inserted between residues L91 and K92 (G i1-91hRLuc) were described previously (Galés et al., 2006). Stables Cell Lines and Transfections. HEK293S cells (Reeves et al., 1996) stably expressing the hemagglutinin-tagged human 1AR, the human -opioid receptor tagged with FLAG ( OR), or the myc-tagged V2 vasopressin receptor (V2R) were described previously (Petaja-Repo et al., 2000; Galandrin and Bouvier, 2006; Charest et al., 2007). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and streptomycin, 2 mM L-glutamine, and 200 g/ml G418, in a 37°C humidified 5% CO2 atmosphere. For all BRET experiments, HEK293 cells were transiently transfected with the indicated BRET partners (tagged receptor and/or tagged G protein subunits) along with the complementary untagged G protein subunits, so to maintain the stoichiometric expression of the G i1 1 2 heterotrimer. For ERK phosphorylation assays, cells were transfected in six-wells plates, and they were harvested 48 h after transfection. In all cases, transient transfections were performed using the FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol. The previously described siRNAs for arrestin 1 and 2 (Ahn et al., 2003) were purchased from QIAGEN and transfected at 400 nM final concentration (300 nM arrestin 1 siRNA and 100 nM arrestin 2 siRNA) using the RNAiFect transfection Reagent (QIAGEN, Mississauga, ON, Canada), according to the manufacturer’s protocol. Quantification of cAMP Accumulation. Agonist induced cAMP accumulation was measured as described previously (Galandrin and Bouvier, 2006). Cells were grown in 60-mm dishes, and they were incubated for 16 h in Dulbecco’s modified Eagle’s medium, with or without 300 ng/ml CTX when indicated. The day of the experiment, cells were resuspended in PBS/0.1% glucose/1 mM 3-isobutyl-1-methylxanthine and treated for 20 min at 37°C with the indicated drugs. Propranolol was tested in the presence of 0.3 M forskolin to increase the signal-to-noise ratio for detecting inhibition, because it has been described as an inverse agonist. After drug treatment, cells were immediately lysed, and cAMP levels were measured using the Catch Point cAMP kit (Molecular Devices, Sunnyvale, CA), according to the manufacturer’s recommendations. In brief, cells lysates were incubated in 384-well plates coated with anti-cAMP antibodies in the presence of known amounts of HRPcAMP. cAMP from cell lysates was allowed to compete with the HRP-cAMP for 2 h, and the remaining peroxidase activity was measured after three washes. The cAMP generated under the different conditions was interpolated from a cAMP standard curve generated in parallel for each experiment. Triplicates were used for each condition, and all experiments were repeated at least three times. Western Blotting. Cells expressing 1AR, OR, or V2R were seeded in poly-D-lysine-coated six-well plates. The next day, cells were washed once with PBS, and they were rendered quiescent by serum starvation for 16 h. Cells ( 80% confluence) were then stimulated at 37°C with conditions corresponding to the maximal response obtained from time course and dose-response experiments: 4 min with 10 M isoproterenol, 2 min with 10 M bucindolol or propranolol, 5 min with 1 M AVP or Leu-enkephalin, or 10 min with 1 ng/ml EGF. To terminate the stimulation, the media were rapidly removed, and cells were placed on ice and washed with ice-cold PBS before being lysed using 100 l/well Laemmli sample buffer (62.5 Ligand-Biased MAPK Signaling at the 1-Adrenegic Receptor 163 at A PE T Jornals on A uust 6, 2017 m oharm .aspeurnals.org D ow nladed from mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue, pH 6.8). Whole cell lysates were sonicated, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The blots were then blocked at room temperature for 1 h with TBS-T buffer [50 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% (v/v) Tween 20] containing 5% fat-free milk. Phospho-ERK1/2 or -ERK2-GFP were detected using mouse polyclonal anti-phospho p42/ p44 ERK-specific antibody (1:3000, overnight at 4°C in TBS-T/5% fat-free milk). The immunoreactivity was revealed using a secondary HRP-conjugated anti-mouse antibody (1:10,000, 1 h at room temperature in TBS-T/5% fat-free milk), and the peroxidase activity was detected by chemiluminescence (PerkinElmer Life and Analytical Sciences, Waltham, MA). Blots were stripped and reprobed for total ERK using rabbit polyclonal anti-ERK1/2 antibody (1:25,000, 1 h at room temperature in TBS-T/5% fat-free milk) followed by HRP-antirabbit antibody (1:20,000, 1 h at room temperature in TBS-T/5% fat-free milk). Films were scanned, and band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA). ERK1/2 or ERK2-GFP phosphorylation was normalized according to the loading of proteins by expressing the data as a percentage of P-ERK1/2/ ERK1/2 total (or P-ERK2-GFP/ERK2-GFP total) of the level observed in agonist-stimulated condition. When using P-ERK2-GFP, because a basal activity was detectable, it was not subtracted from the ligand-promoted ERK activity. Phosphorylation of PKA substrates was revealed using a rabbit polyclonal anti-P-SPKA antibody, detecting proteins containing a phosphor-Ser/Thr residue with arginine at the 3 position (1:1000, overnight at 4°C in TBS-T/5% bovine serum albumin), followed by anti-rabbit HRP-conjugated IgG (1:10,000, 1 h at room temperature in TBS-T/5% fat-free milk). Detection of arrestin1 and -2 was achieved using the mouse monoclonal antiarrestin2 (H9) antibody, which recognizes both arrestin isoforms (1:1000, overnight at 4°C in TBS-T/0.5% fat-free milk), followed by anti-mouse HRP-conjugated IgG (1:5000, 1 h at room temperature in TBS-T/0.5% fat-free milk). Bioluminescence Resonance Energy Transfer Measurement. Rlucand GFP10-tagged receptor or G protein constructs were transiently transfected into HEK293 cells. Forty-eight hours after transfection, cells were washed twice with PBS, detached with PBS/5 mM EDTA, and resuspended in PBS/0.1% (w/v) glucose at room temperature. Cells were then distributed (50 g of protein per well) in a 96-well microplate (Optiplate; PerkinElmer Life and Analytical Sciences) and incubated in the presence or absence of the different ligands for 1 min. BRET between Rluc and GFP10 was measured after the addition of the Rluc substrate DeepBlueC coelenterazine (5 M) (PerkinElmer Life and Analytical Sciences). BRET readings were collected using a modified TopCount NXT apparatus (customized version purchased from BioSignal Packard, Inc., now; PerkinElmer Life and Analytical Sciences) that allows sequential integration of signals detected in the 370to 450and 500to 530-nm windows, using filters with the appropriate band pass (Chroma Technology Corp., Brattleboro, VT). The BRET signal was calculated as the ratio of the light emitted by GFP10 (510–550 nm) over the light emitted by Rluc (460–500 nm). Statistical Analysis. Statistical analysis and curve fitting were done using Prism 3.01 (GraphPad Software, San Diego, CA). Statistical significance of the differences was assessed using one-way analysis of variance and post hoc Bonferroni’s test. In some cases, oneway analysis of variance followed by Dunnett’s test was used to determine statistically significant differences from basal.