Factors Influencing Biased Agonism in Recombinant Cells Expressing The

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British Journal of British Journal of Pharmacology (2017) 174 2318–2333 2318 BJP Pharmacology RESEARCH PAPER Factors inﬂuencing biased agonism in recombinant cells expressing the human α1A-adrenoceptor

Correspondence Roger J Summers and Bronwyn A Evans, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia. E-mail: [email protected]; [email protected]

Received 1 February 2017; Revised 6 April 2017; Accepted 12 April 2017

Edilson Dantas da Silva Junior, Masaaki Sato, Jon Merlin, Natalie Broxton, Dana S Hutchinson, Sabatino Ventura, Bronwyn A Evans and Roger J Summers

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia

BACKGROUND AND PURPOSE Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have α examined bias at human 1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. EXPERIMENTAL APPROACH Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. KEY RESULTS Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efﬁcacy for Ca+2 mobilization, no effect on cAMP generation and high efﬁcacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off- target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. CONCLUSION AND IMPLICATIONS α We have shown that while adrenergic agonists display bias at human 1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

Abbreviations ECAR, extracellular acidiﬁcation rate; HEAT, 2-(β-4-hydroxyphenyl)ethylaminomethyltetralone;PTX,pertussistoxin

Introduction Artinano, 2001) including the vas deferens (Campos et al., 2003), seminal vesicle (Silva et al., 1999), cauda epididymis Agonists acting at GPCRs are often shown to promote (Queiroz et al., 2002) and prostate gland (Gray et al., 2008). promiscuous receptor coupling and to activate multiple This uroselectivity has driven the development of selective signalling pathways (Kukkonen et al., 2001; Perez and Karnik, drugs for the treatment of benign prostatic hypertrophy 2005; Kenakin and Christopoulos, 2013). Some agonists can (Ventura et al., 2011), and this receptor is also a target for male α selectively activate signalling pathways by stabilizing distinct contraception (White et al., 2013). It is now evident that 1A- receptor conformations, thereby promoting differential adrenoceptors have important metabolic (Hutchinson and interaction with particular Gα subunits or alternative effector Bengtsson, 2005; Hutchinson and Bengtsson, 2006) and proteins, a characteristic known as biased agonism or cardioprotective effects (O’Connell et al., 2014). α α functional selectivity (Evans et al., 2010; Kenakin and The 1A-adrenoceptor classically activates G q/11, Christopoulos, 2013; Luttrell et al., 2015). Biased agonists stimulating PLC to hydrolyse phosphatidylinositol that promote beneficial cellular responses but not 4,5-bisphosphate and form inositol 1,4,5-trisphosphate and detrimental effects are considered to have great therapeutic diacylglycerol. These molecules mediate intracellular Ca2+ promise. Biased agonism has been studied primarily in mobilization and activation of PKC, respectively (Chen and α recombinant GPCR expression systems with the advantages Minneman, 2005). 1A-adrenoceptors also activate multiple that receptor abundance can be modulated, and nominally signalling pathways in addition to canonical effects on Ca2+ α that only the receptor subtype of interest is present. It is mobilization. For instance, activation of 1A-adrenoceptors essential, however, that studies performed on cells expressing mayincreasecAMPaccumulationandERK1/ERK2 recombinant GPCRs represent bias that can be translated to phosphorylation in native tissues and in recombinant therapeutically relevant cells, tissues and in vivo experiments. systems (Ruan et al., 1998; Hu et al., 1999; Jiao et al., 2002; For example, the agonist TRV130 (oliceridine) displays Bauer et al., 2011; Evans et al., 2011; Perez-Aso et al., 2013). marked bias towards Gαi/o-mediated inhibition of cAMP Agonist bias towards particular signalling pathways has also accumulation over β-arrestin recruitment in HEK293 cells been demonstrated for different ligands and we have μ α expressing the -opioid receptor from human and other previously shown that phenylephrine ( 1-adrenoceptor mammalian species (DeWire et al., 2013). In animal studies, agonist) displays substantial bias towards extracellular oliceridine was found to have analgesic potency similar to acidification rate (ECAR) versus Ca2+ mobilization or cAMP α morphine, but produced less gastrointestinal dysfunction accumulation. On the other hand, A61603 ( 1A- and respiratory depression than morphine. Recent Phase 3 adrenoceptor agonist) displays bias towards cAMP clinical trials have demonstrated effective post-operative accumulation relative to Ca2+ mobilization while analgesia following oliceridine treatment, with diminished oxymetazoline showsbiastowardsECARcomparedto nausea and vomiting compared to morphine. Another Ca2+ in CHO-K1 cells over-expressing the human κ α example is the development of biased agonists at the -opioid 1A-adrenoceptor (Evans et al., 2011). receptor to treat pain and chronic itch, but lacking the Here,wehaveinvestigatedbiasedagonisminCHO-K1 α adverse effects of dysphoria and sedation (Brust et al., 2016). cells expressing low levels of the human 1A-adrenoceptor κ α 1 The -receptor agonist triazole 1.1 displayed bias towards (CHO 1A-adrenoceptor, 204 fmol·mg protein). All of the beneficial Gαi/o signalling over detrimental β-arrestin agonists except oxymetazoline increased intracellular Ca2+ mediated pathways. This bias in CHO-K1 cells expressing mobilization, cAMP accumulation and ERK1/2 the κ-receptor (Zhou et al., 2013) was reflected in mouse phosphorylation. Oxymetazoline was a partial agonist for behavioural studies showing that triazole 1.1 had Ca2+ mobilization and a super-agonist for ERK1/2 antinociceptive and antipruritic activity without β-arrestin- phosphorylation yet failed to stimulate cAMP accumulation mediated dopaminergic inhibition (Brust et al., 2016). even at high concentrations. We found that phenylephrine, Despite these successes, the determination of relevant methoxamine and A61603 displayed ligand bias at α fi agonist bias remains a challenge, and many factors can 1A-adrenoceptors. However, the distinct signalling pro le confound interpretation such as off-target effects, cell of oxymetazoline, particularly for ERK1/2 phosphorylation, background, receptor and effector expression levels, and the was related to off-target effects at 5-HT1B receptorsthat kinetic properties of agonists. Thus, while recombinant cell are endogenously expressed in CHO-K1 cells. lines are generally checked for expression of related receptor subtypes from the same family as the target GPCR, less attention has been paid to the possibility that biased agonists may be acting at both the target receptor and another Methods receptor belonging to a distinct GPCR family that is expressed endogenously in the host cells. This type of ‘off-target’ Cell culture and production of clones stably activity is likely to be widespread, based on classification of expressing α1A-adrenoceptors α receptors by the chemical similarity of their ligands (Keiser Plasmid containing the human 1A-4-adrenoceptor cDNA et al., 2009; Lin et al., 2013). Our study examines this problem was kindly donated by Dr. Thomas Chang (Roche α fi in CHO-K1 cells expressing the human 1A-adrenoceptor. Bioscience, Palo Alto, CA) and modi ed to produce the α α 1A-adrenoceptors are the main adrenoceptor subtype human 1A-1-adrenoceptor as described by Evans et al. involved in the contractile responses to endogenous or (2011). CHO-K1 cells were cultivated in a 50:50 DMEM exogenous agonists in the smooth muscle of the /Ham’s F12 medium supplemented with 5% (v.v 1)FBS, genitourinary system (Civantos Calzada and Aleixandre de glutamine (2 mM), penicillin (100 U mL 1)and

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μ 1 1 streptomycin (100 g·mL ) at 37°C with 5% CO2. probenecid, pH 7.4) containing 0.5% (w·v ) BSA. In light- Transfections were performed using Lipofectamine diminished conditions, cells were treated with fluoro-4 (Invitrogen) as per the manufacturer’sinstructions. (0.1% (v·v 1)inmodified HBSS for 1 h at 37°C. Cells were Transfected cells were selected in media containing washed twice in modified HBSS and incubated for a further G418 (800 μg·mL 1) and maintained in media containing 30 min before transfer to a FlexStation (Molecular Devices, 400 μg·mL 1 G418. Clonal cell lines were isolated by Sunnyvale, CA). Real-time fluorescence measurements were selecting G418-resistant colonies and preliminary screening recorded every 1.7 over 200 s, with drug additions occurring of clones made using 2000 pM of [3H]-prazosin in after17s,atanexcitationwavelengthof485nmand whole-cell binding assays. Suitable clones were amplified emission wavelength of 520 nm. All experiments were and receptor levels determined in saturation binding assays performed in duplicate. Agonist responses represent the 125 fl 2+ using [ I]-HEAT. Transient transfection of HEK293 cells difference between basal uorescence and peak [Ca ]i α with the 1A-adrenoceptor construct in pcDNA3.1 was measurements expressed as a percentage of the response to performed using polyethylenimine (PEI). Transfection the Ca2+ ionophore A23187 (1 μM) in each experiment. solution comprised NaCl (150 mM; pH 7.0) containing 15 μL PEI (1 mg·mL 1)and5μg DNA incubated at room cAMP accumulation or inhibition studies temperaturefor20minwith200μL added to each well of a Cells were seeded into 96-well plates at 25000 cells per well in six-well plate containing 500 000 cells in growth medium. media with 0.5% (v·v 1) FBS the night before the experiment. Transfected cells were harvested after 24 h and plated into On the day of the experiment, the media was replaced with 96-well plates for 6 h before serum starving overnight prior stimulation buffer (1 mg·mL 1 BSA, 0.5 mM IBMX, and to experimentation. 5 mM HEPES, pH 7.4, in HBSS, 90 μL per well). Cells were exposed to agonists for 30 min at 37°C in cAMP accumulation Radioligand binding studies assays. For the study evaluating the inhibition of cAMP production, cells were treated with oxymetazoline or 5-HT Cells from a 75 cm2 flask were washed twice with HEPES- for 15 min, then forskolin (1 μM) was added for 30 min. buffered saline and scraped from flasks with lysis buffer The reactions were terminated by the removal of the (25 mM Tris, pH 7.5 at room temperature (21°C), 1 mM stimulation buffer and the addition of 50 μL ice-cold 100% EDTA, 10 μg·mL 1 leupeptin, 200 μg·mL 1 bacitracin, 2.5- ethanol. The uncovered plates were then incubated at 37°C, μg·mL 1 aprotinin and 2.5 μg·mL 1 pepstatin A,). The to allow complete evaporation of the ethanol. After the suspension was homogenised in a Dounce homogeniser and ethanol was evaporated, 50 μL of detection buffer [1 mg·mL 1 centrifuged at low speed (1000 g, 10 min) to remove cell BSA, 0.3% (v.v 1) Tween 20 and 5 mM HEPES, pH 7.4] was debris. Supernatants were pooled and centrifuged (39 000 g, added to each well. cAMP accumulation was measured using 15 min, 4°C). The pellet was resuspended in binding buffer ™ μ 1 aLance cAMP kit (PerkinElmer), and detected using the (50mMTris,pH7.4,5mMMgCl2,1mMEDTA,200 g·mL ™ EnVision Multilabel Plate Reader (PerkinElmer). Results are bacitracin, 10 μg·mL 1 leupeptin, 2.5 μg·mL 1 pepstatin A, expressed as the percentage of the forskolin response and 2.5 μg·mL 1 aprotinin) and frozen at 70°C until (100 μM) in the given experiment. All experiments were required. Membranes (10–20 μgofprotein)wereincubated performed in duplicate, and n is the number of independent with [125I]-HEAT (20–1200 pM) in a total volume of 100 μL experiments. for 90 min at room temperature (21°C). Phentolamine μ fi fi (100 M) was used to de ne nonspeci cbinding. AlphaScreen ERK1/2 signalling assays Competition binding experiments were conducted using a ERK1/2 phosphorylation was detected using the AlphaScreen range of concentrations of unlabelled ligand and [125I]-HEAT SureFire Assay Kit (PerkinElmer Life Sciences). Briefly, cells (500 pM), at room temperature for 90 min (Sharpe et al., were seeded in clear 96-well plates (10 000 cells per well) 2003). Reactions were terminated by rapid filtration through and allowed to adhere for 24 h, then incubated in serum-free GF/C filters presoaked for 30 min in 0.5% polyethylenimine DMEM/ Ham’s F12 medium overnight at 37°C and 5% CO . using a Packard Filtermate cell harvester. Filters were washed 2 Cellsweretreatedinthepresenceorabsenceofantagonists three times with wash buffer (50 mM Tris, pH 7.4, 4 °C), for 30 min (or 12–18 h for PTX), before stimulation with dried, and 25 μL of Microscint O (PerkinElmer Life and agonists for 5 min. The medium was replaced with SureFire Analytical Sciences) added and radioactivity counted on a lysis buffer (TGR Biosciences) followed by incubation for Packard TopCount. Protein concentrations were quantified 5 min at room temperature with agitation, and samples were using the Lowry et al. (1951) assay. All experiments were frozen at 20°C. Approximately 5 μL of lysate per well was performed in duplicate using at least three different transferred to a 384-well white ProxiPlate (PerkinElmer), membrane preparations. and 4 μL of acceptor mix (activation buffer, reaction buffer, and AlphaScreen beads (10:40:1) added. Plates were 2+ Measurement of intracellular free Ca incubated for 2 h at 37°C before addition of 2 μL of donor concentration mix (donor beads and dilution buffer, 1:20), then incubated α ™ CHO 1A-adrenoceptor cells were seeded at 25000 cells per for 2 h at 37°C. Plates were read on an EnVision Multilabel well in 96-well plates overnight. On the day of the Plate Reader (PerkinElmer). experiment, the medium was removed and cells washed three times in a modified Hanks’ balanced saline solution (HBSS; TaqMan RTand real time qPCR α 150mMNaCl,2.6mMKCl,1.18mMMgCl2.2H2O, 10 mM RNA was extracted from CHO 1A-adrenoceptor cells and from D-glucose, 10 mM HEPES, 2.2 mM CaCl2.2H2O, and 2 mM untransfected CHO-K1 cells. Total RNA was isolated using

2320 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP

TRIzol reagent (Invitrogen Life Technologies, Inc.) according 5,6,7,8-tetrahydro naphthalen-1-yl] methanesulphonamide to the manufacturer’s protocol. RNA quality was assessed by hydrobromide), serotonin creatinine sulfate monohydrate absorbance at 260 and 280 nm and electrophoresis on 1.3% (5-HT), prazosin hydrochloride, calcimycin (A23187) and agarose gels. Degradation of RNA samples was not detected. pertussis toxin (PTX) were purchased from Sigma-Aldrich ™ DNase (DNA-free DNA removal kit, Invitrogen Life (St Louis, MO). SB216641 (N-[3-[3-(dimethylamino) Technologies, Inc.) was added to remove DNA ethoxy]-4-methoxyphenyl]-20-methyl-40-(5-methyl-1,2,4- contamination. cDNAs were synthesised by RT following oxadiazol-3-yl)-[1,10-biphenyl]-4-carboxamide ™ the manufacturer’s protocol (iScript Reverse Transcription hydrochloride) was purchased from Tocris Bioscience Supermix for RT-qPCR, BioRad). Briefly, reaction mix (Bristol, UK); UBO-QIC (name since revised to (10 μL) containing 500 ng of RNA was incubated in a FR900359) was acquired from the Institute of thermal cycler through priming (5 min at 85°C), RT Pharmaceutical Biology, University of Bonn, Germany. (30 min at 42°C) and RT inactivation (5 min at 85°C) cycles. RT reactions were diluted 1 in 20 and 4 μLofcDNAusedin conjunctionwithTaqManGeneExpressionAssaysto Nomenclature of targets and ligands Key protein targets and ligands in this article are measure 5-HT1B receptor (Chinese hamster hyperlinked to corresponding entries in http://www. Cg04423773_s1) or Actb expression (Cg04424027_gH, guidetopharmacology.org, the common portal for data from Invitrogen Life Technologies, Inc). Samples were amplified the IUPHAR/BPS Guide to PHARMACOLOGY (Southan in a Bio-Rad CFX96 instrument according to the following et al., 2016), and are permanently archived in the Concise protocol: uracil-N glycosylase incubation (2 min at 50°C) Guide to PHARMACOLOGY 2015/16 (Alexander et al., and AmpliTaq Gold activation (10 min at 95°C), PCR 2015a,b). (40 cycles), each cycle consisting of denaturation (15 s at 95°C) followed by annealing and extension (1 min at 60°C). All TaqMan assays were performed in duplicate. Levels of Htr1b mRNA are expressed relative to Actb, Results Δ calculated as (2- Ct)*1000. Expression of α1A-adrenoceptors in CHO-K1 Data analysis cells 125 All values are expressed as mean ± SEM. Data were analysed Saturation binding studies with [ I]-HEAT revealed a Bmax 1 using nonlinear curve fitting (Prism ver. 6.0; GraphPad of 204 ± 28 fmol·mg (n =7)proteinandpKD of 2+ Software, San Diego, CA) to obtain pEC50 values for Ca 9.3±0.06(n = 7) similar to those reported for α 1 mobilization, cAMP accumulation, ECAR and ERK1/2 1-adrenoceptors in rat brain cortex (Bmax 116 fmol·mg ) 1 phosphorylation, or pKi,pKD,andBmax values from (Graziadei et al., 1989), bovine aorta (Bmax 170 fmol·mg ) radioligand binding assays. The statistical significance of (Descombes and Stoclet, 1985) or rabbit prostate (Bmax 1 differences between groups was analysed by one-way ANOVA 210 fmol·mg )(Suet al., 2008). Competition studies with Bonferroni post test correction or Student’sunpaired with noradrenaline, phenylephrine, methoxamine, A61603 two-tailed t-test (*P < 0.05). Data are presented as the increase and oxymetazoline were also carried out and showed that 125 expressed as a % of basal levels, if not otherwise stated. all agonists competed for [ I]-HEAT binding with curves Agonist bias was quantified as previously reported (Evans fitting a single-site isotherm. The rank order of binding fl τ fi et al., 2011; Kenakin et al., 2012). Brie y, the Log( /KA)or af nities (pKi values) was: oxymetazoline (7.80 ± 0.09, LogTR value of a reference agonist, in this case noradrenaline, n = 3) = A61603 (7.53 ± 0.18, n =3)≫ noradrenaline is subtracted from the LogTR value of the agonists of interest (5.57 ± 0.09, n =3)≥ phenylephrine (5.30 ± 0.06, > to yield LogTRn in order to avoid the impact of cell- and assay- n =3) methoxamine (4.60 ± 0.12, n =3). dependent effects on the observed agonism in each pathway. The relative bias is then calculated for each agonist at the two distinct signalling pathways by subtracting the LogTRn of one Signalling responses to adrenergic agonists in Δ α pathway from the other to give a LogTRn value, a numerical CHO cells expressing 1A-adrenoceptors measure of bias. A lack of biased agonism will result in values Concentration response curves for phenethylamines Δ fi of LogTRn not signi cantly different from 0. The data and (noradrenaline, phenylephrine and methoxamine) and statistical analysis comply with the recommendations on imidazolines (A61603 and oxymetazoline) were performed for experimental design and analysis in pharmacology (Curtis Ca2+ mobilisation (Figure 1A), cAMP accumulation (Figure 1B) et al., 2015). and ERK1/2 phosphorylation (p-ERK1/2) (Figure 1C) in α CHO 1A-adrenoceptor cells. All phenethylamines and Materials imidazolines increased Ca2+ mobilisation, cAMP accumulation [125I]-HEAT (2-[β-(4-hydroxy-3-[125I]iodophenyl) ethylamino- and ERK1/2 phosphorylation (Figure 1). However, methyl]tetralone) was purchased from Pro-Search, oxymetazoline was a weak partial agonist (α = 0.35) for Melbourne, Australia. [3H]prazosin was obtained from Ca2+ mobilization (Table 1), failed to significantly affect PerkinElmer Life and Analytical Sciences, Waltham, MA. cAMP accumulation, but was a super agonist (α = 2.3) for (-)-Norepinephrine bitartrate (noradrenaline), ERK1/2 phosphorylation (Table 1). The order of potency oxymetazoline hydrochloride, phenylephrine for Ca2+ mobilization was A61603 = oxymetazoline > hydrochloride, methoxamine hydrochloride, A61603 noradrenaline = phenylephrine = methoxamine; for cAMP hydrate (N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy- accumulation, A61603 > noradrenaline = phenylephrine >

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Figure 1 Concentration-response curves for Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation (p-ERK1/2) in CHO-K1 cells expressing α 2+ human 1A-adrenoceptors. Concentration-dependent Ca mobilization (A), cAMP accumulation (B) or ERK1/2 phosphorylation (C) was stimulated by the phenethylamines noradrenaline, phenylephrine and methoxamine, and the imidazolines A61603 and oxymetazoline. Each point represents mean ± SEM of n (in brackets) independent experiments. *P<0.05 in relation to the maximum response of agonists other than oxymetazoline.

Table 1 α α Potency (pEC50), maximum response (Emax) and value (Emax relative to noradrenaline) of agonists at the human 1A-adrenoceptors across different signalling outcomes. Ca2+ mobilization was measured as % 23187(1 μM), cAMP accumulation as % forskolin (100 μM) and pERK1/2 as fold/basal.

Agonists Parameters Ca2+ cAMP p-ERK1/2

Noradrenaline Emax 47 ± 1.8 (8) 11.7 ± 0.45 (6) 2.0 ± 0.5 (15)

pEC50 7.7 ± 0.22 5.8 ± 0.13 6.3 ± 0.12 α 1.00 1.00 1.00

Phenylephrine Emax 38.1 ± 1.7 (8) 7.9 ± 0.40 (6) 1.54 ± 0.4 (8)

pEC50 7.0 ± 0.18 5.3 ± 0.16 6.6 ± 0.3 α 0.81 0.67 0.77

Methoxamine Emax 34.4 ± 1.7 (8) 7.8 ± 0.44 (6) 1.7 ± 0.09 (8)

pEC50 6.4 ± 0.16 4.6 ± 0.15 7.1 ± 0.43 α 0.73 0.67 0.85

A61603 Emax 51.3 ± 2.0 (8) 12.7 ± 0.49 (6) 2.0 ± 0.08 (14)

pEC50 9.5 ± 0.17 7.8 ± 0.13 7.45 ± 0.22 α 1.1 1.1 1.0

Oxymetazoline Emax 16.6 ± 0.9 (8) N.D. 4.6 ± 0.13 (17)

pEC50 9.3 ± 0.36 N.D. 7.2 ± 0.10 α 0.35 N.D 2.3 Values are mean ± SEM of n (in brackets) independent experiments N.D.: not determined methoxamine >>oxymetazoline; and for p-ERK1/2, of distinct signalling pathways after occupying > α A61603 = oxymetazoline = methoxamine phenylephrine = 1A-adrenoceptors (indicating functional selectivity) or to noradrenaline (see Table 1 for pEC50 values). Moreover, off-target effects. phenylephrine and methoxamine produced lower Emax values than the reference agonist noradrenaline in all signalling assays (Table 1) and A61603 behaved as a full Characterization of ERK1/2 phosphorylation agonist (compared to noradrenaline) (Figure 1). The unique induced by oxymetazoline and other adrenergic and markedly biased behaviour of oxymetazoline was agonists α further evaluated to determine whether its differential Prazosin ( 1-adrenoceptor antagonist) (300 nM), was used to α effects on ERK1/2 phosphorylation were due to activation examine the contribution of 1A-adrenoceptors to ERK1/2

2322 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP phosphorylation mediated by oxymetazoline. The same (Figure 2C). Together, these data suggested that protocol was used for noradrenaline (reference agonist) and oxymetazoline increases ERK1/2 phosphorylation by

A61603 (an imidazoline highly selective for α1A- activating a receptor other than the α1A-adrenoceptor or by α adrenoceptors). Prazosin produced a rightward shift binding to a site on the 1A-adrenoceptor distinct from that (approximately 10-fold) of the concentration–response identiﬁed by noradrenaline. The maximum response curves to noradrenaline (Figure 2A) and A61603 (Figure 2B), produced by noradrenaline and A61603 was inhibited by α α indicating involvement of 1A-adrenoceptors but was much pre-incubation with UBO-QIC (100 nM) (G q inhibitor), less effective in antagonizing responses to oxymetazoline whereas that to oxymetazoline was unaffected (Figure 2D–F).

Figure 2

Effect of the α1-adrenoceptor antagonist prazosin, the Gq inhibitor UBO-QIC or the Gi/o inhibitor PTX on ERK1/2 phosphorylation (p-ERK1/2) α produced by noradrenaline, A61603 and oxymetazoline in CHO-K1 cells expressing human 1A-adrenoceptors. Cells were stimulated for 5 min in the presence of increasing concentrations of noradrenaline, A61603 and oxymetazoline in the absence or presence of prazosin 300 nM (pre-incubated for 30 min; A–C), UBO-QIC 100 nM (pre-incubated for 30 min; D–F) or PTX 1 ng·mL 1 (pre-incubated for 12–18 h; G–I). Each point represents mean ± SEM from n (in brackets) independent experiments. *P < 0.05 in relation to the control maximum response.

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PTX (1 ng·mL 1)(Gαi/o inhibitor) pretreatment did not 2012). The CHO-K1 transcriptome is available on NCBI Gene inhibit ERK1/2 phosphorylation to A61603 (Figure 2G), Expression Omnibus (Accession number GSE75521). Table S1 slightly enhanced the maximum response to noradrenaline provides expression values for all GPCRs common to the (Figure 2H) but abolished the response to oxymetazoline CHO-K1 sequence data. fi α (Figure 2I). Importantly, we found that oxymetazoline Oxymetazoline is a high-af nity agonist at both 1A- α produced robust ERK1/2 phosphorylation in untransfected adrenoceptor and 2A-adrenoceptors (pEC50 values of 8.2 CHO-K1 cells (Figure 3B). and 8.6 respectively) (Uhlen et al., 1994), but lower affinity for all remaining α-adrenoceptors (pKi 6.2–6.7). It also has fi high af nity for 5-HT1B and 5-HT1D receptors (pKi 9.5 and α Theroleof5-HT1B receptors in ERK1/2 9.4, Law et al., 1998), and acts at G i/o-coupled 5-HT1B phosphorylation stimulated by oxymetazoline receptors to inhibit forskolin-stimulated adenylate cyclase fi We identi ed the 5-HT1B receptor as an alternative target activity (Schoeffter and Hoyer, 1991). The CHO-K1 RNA for oxymetazoline (http://www.guidetopharmacology.org/ sequence data indicated that the 5-HT1B receptor is expressed α download.jsp) in conjunction with publically available but not the 5-HT1D receptor, 2A-adrenoceptor or other RNA sequencing data for CHO-K1 cells (Baycin-Hizal et al., α-adrenoceptors (Table S1). Since earlier studies show that

Figure 3

ERK1/2 phosphorylation (p-ERK1/2) produced by 5-HT, oxymetazoline, noradrenaline, A61603 in CHO-K1 cells expressing human α1A- α adrenoceptors (AR) or untransfected CHO-K1 cells. In (A) CHO-K1 cells expressing human 1A-adrenoceptors were stimulated for 5 min in the presence of increasing concentrations of 5-HT, noradrenaline, A61603 and oxymetazoline. In (B) untransfected CHO-K1 cells were stimulated for 5 min in the presence of increasing concentrations of 5-HT, noradrenaline, A61603 and oxymetazoline. (C) Comparison of the maximum response (Emax) of agonists in CHO-K1 cells expressing human α1A-adrenoceptors or untransfected CHO-K1 cells. n values in brackets; P < 0.05 in relation to the maximum response of *noradrenaline or #oxymetazoline in CHO-K1 cells expressing human α1A-adrenoceptors. (D) Transcript α levels of 5-HT1B receptors in CHO-K1 cells expressing human 1A-adrenoceptors or untransfected CHO-K1 cells.

2324 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP

CHO-K1 cells endogenously express low densities of 5-HT1B (Figure 5C). The potency of oxymetazoline was decreased receptors (Giles et al., 1996), we conducted functional studies approximately ninefold by prazosin plus SB216641 (Figure 5 to examine their contribution to oxymetazoline responses. D). Together with the UBO-QIC and PTX inhibition, this α 5-HT increased ERK1/2 phosphorylation with similar indicated that oxymetazoline interacts with both 1A- fi α potency and ef cacy to oxymetazoline in CHO 1A- adrenoceptors and 5-HT1B receptors, with the net effect on adrenoceptor cells (Figure 3A) and also in untransfected ERK1/2 phosphorylation due primarily to the activation of α CHO-K1 cells (Figure 3B). In contrast, in untransfected 5-HT1B receptors. The response to other 1A-adrenoceptor CHO-K1 cells, noradrenaline, A61603, methoxamine or agonistsmaybemodulatedbythepresenceof5-HT1B phenylephrine did not increase ERK1/2 phosphorylation receptors in CHO-K1 cells, as SB216641 decreased maximum (Figure 3C, Supporting Information Figure S1). We verified ERK1/2 phosphorylation in response to noradrenaline and using real time qPCR that transcripts for 5-HT1B receptors A61603 (Figure 5A,B), while prazosin had low potency in α are present in both untransfected CHO-K1 and in CHO 1A- antagonizing ERK1/2 responses to these two agonists adrenoceptor cells (Figure 3D). Concentration response (Figure 2A,B). curves to 5-HT were right-shifted 10-fold by SB216641 ERK1/2 phosphorylation in HEK293 cells was also α (5-HT1B antagonist; 300 nM) (Figure 4A) whereas prazosin measured in response to the 1-adrenoceptor agonists and was ineffective (Figure 4B). ERK1/2 phosphorylation to 5-HT 5-HT (Supporting Information Figure S3), since these cells fi α was unaffected by UBO-QIC (Figure 4C) but signi cantly do not express 5-HT1B or 1A-adrenoceptors (Supporting decreased by pretreatment with 1 ng·mL 1 PTX (Figure 4D). Information Table S1). HEK293 cells showed no ERK1/2 SB216641 (300 nM) reduced the potency of response to A61603, oxymetazoline, phenylephrine or 5-HT α oxymetazoline 3.5-fold and the maximum response by ~30% whereas cells transiently transfected with 1A-adrenoceptor

Figure 4

Effect of the 5-HT1B antagonist SB216641, the α1-adrenoceptor antagonist prazosin, the Gq-inhibitor UBO-QIC, or the Gi/o-inhibitor PTX on α ERK1/2 phosphorylation produced by 5-HT in CHO-K1 cells expressing human 1A-adrenoceptors. Cells were stimulated for 5 min in the presence of increasing concentrations of 5-HT in the absence or presence of SB216641 (300 nM, pre-incubated for 30 min; A), prazosin (300 nM, pre- incubated for 30 min; B), UBO-QIC (100 nM, pre-incubated for 30 min; C) or PTX (1 ng.mL-1, pre-incubated for 12–18 h; D). Each point represents mean ± SEM of n (in brackets) independent experiments. *P < 0.05 in relation to the maximum response of the control.

British Journal of Pharmacology (2017) 174 2318–2333 2325 E D da Silva Junior et al. BJP

Figure 5

Effect of the 5-HT1B antagonist SB216641 on ERK1/2 phosphorylation produced by noradrenaline, A61603 or oxymetazoline in CHO-K1 cells expressing human α1A-adrenoceptors. Cells were stimulated for 5 min in the presence of increasing concentrations of agonist in the absence or presence of SB216641 (300 nM, pre-incubated for 30 min; A–C). In (D) the effect of SB216641 (300 nM, pre-incubated for 30 min), prazosin (300 nM, pre-incubated for 30 min) or SB216641 plus prazosin on oxymetazoline-induced p-ERK1/2. Each point represents mean ± SEM of n (in brackets) independent experiments. *P < 0.05 in relation to the maximum response of the control. # P < 0.05 in relation to the maximum response of oxymetazoline curve in the presence of prazosin. showed robust ERK1/2 phosphorylation in response to Oxymetazoline promotes cAMP accumulation A61603, with smaller responses to methoxamine, inthepresenceof1μM forskolin but not phenylephrine and 5-HT. Oxymetazoline was a weak partial following pretreatment of CHOα1A- agonist for ERK1/2 phosphorylation in HEK293 cells whereas adrenoceptor cells with PTX noradrenaline increased ERK1/2 phosphorylation in both We next examined whether the inability of oxymetazoline to parental and transfected HEK293 cells. increase cAMP accumulation is due to the simultaneous α activation of endogenous G i/o-coupled 5-HT1B receptors 2+ α α Ca mobilization and cAMP accumulation are and G q-coupled 1A-adrenoceptors, by examining not stimulated by adrenergic agonists in inhibition of forskolin-induced cAMP accumulation by α untransfected CHO-K1 cells oxymetazoline and 5-HT in untransfected and in CHO 1A- Ca2+ mobilization (Supporting Information Figure S2A) or adrenoceptor cells. 5-HT inhibited cAMP accumulation to μ α cAMP accumulation (Supporting Information Figure S2B) forskolin (1 M) in both cell types (pEC50 CHO 1A- were not increased by noradrenaline, methoxamine, adrenoceptor cells: 8.6 ± 0.44; pEC50 untransfected CHO-K1 phenylephrine and A61603, or by 5-HT (Supporting cells: 8.9 ± 0.33) (Figure 6A,B). Oxymetazoline inhibited Information Figure S2E,F) in untransfected CHO-K1 cells. forskolin-induced cAMP accumulation only in untransfected 2+ α 5-HT also failed to affect Ca mobilization or cAMP CHO-K1 cells (pEC50: 8.9 ± 0.46) whereas in CHO 1A- α accumulation in CHO 1A-adrenoceptor cells (Supporting adrenoceptor cells, oxymetazoline promoted a robust Information Figure S2C,D). increase in cAMP accumulation in the presence (pEC50:

2326 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP

Figure 6

Effects of oxymetazoline and 5-HT on forskolin-stimulated cAMP accumulation in CHO-K1 cells expressing human α1A-adrenoceptors (AR) or α untransfected CHO cells. CHO-K1- 1A-adrenoceptor cells (A) or untransfected CHO cells (B) were stimulated for 15 min in the presence of increasing concentrations of oxymetazoline or 5-HT and then incubated with forskolin (1 μM) for 30 min. CHO-K1-α1A-adrenoceptor cells (C) were also stimulated with oxymetazoline for 30 min in the absence or presence of PTX (1 ng.mL-1 pre-incubated for 12-18 h). Each point represents mean ± SEM of n (in brackets) independent experiments.

8.1 ± 0.40; Figure 6A,B) but not in the absence of forskolin, Agonist bias at α1A-adrenoceptors stably even following pretreatment with PTX. This suggests that expressed in CHO-K1 cells α oxymetazoline-stimulated coupling of endogenously Signalling bias at 1A-adrenoceptors displayed by α expressed 5HT1B receptors to G i/o proteins does not inhibit noradrenaline, phenylephrine, methoxamine and A61603 thecAMPresponse(Figure6C). was quantiﬁed (Figure 7). Oxymetazoline was excluded due

Figure 7 Δ Ligand bias ( LogTRn) across different signalling pathways compared to the reference agonist, noradrenaline. The LogTR values were normalized to that of noradrenaline at each pathway (LogTRn) (Supporting Information Table S2) and compared across different signalling pathways to give a ΔLogTRn (ligand bias) value. *P < 0.05 in relation to the reference agonist noradrenaline. Student’s t-tests were performed on the raw LogTR data (Supporting Information Table S3) to avoid propagation of error. Values expressed as mean ± SEM of n independent experiments (Figure 1).

British Journal of Pharmacology (2017) 174 2318–2333 2327 E D da Silva Junior et al. BJP to the small cAMP response (Figure 1B) and off-target effects investigation. If this is not the case, two lines of evidence (Figure 1C). Concentration–response data (Figure 1) was used should be sought. RNA sequencing data for commonly used to determine ligand bias, and the operational model used to cell lines is now more widely available, and can be used to fi τ derive a transduction coef cient ( /KA or TR) for each agonist assess the presence of GPCR mRNAs in host cells (Uhlen at each pathway (Supporting Information Table S2). LogTR et al., 2015). While there is not always a close correlation values were then normalized to noradrenaline at each between steady-state mRNA and protein levels, the presence pathway (LogTRn) (Supporting Information Table S2) and of receptor expression is an important pointer for further compared across different signalling pathways to give a studies (Supporting Information Table S1). Complementary Δ LogTRn (agonist bias) value (Figure 7). Lack of biased evidence can be found in the IUPHAR Guide to agonism compared to the reference agonist (noradrenaline) Pharmacology (http://www.guidetopharmacology.org), the Δ resultsinvaluesof LogTRn of 0 between pathways Psychoactive Drug Screening Program Ki database (https:// (Student’s t-tests on raw LogTR data rather than data kidbdev.med.unc.edu/databases/ kidb.php) and the literature normalized to norepinephrine as shown in Supporting for off-target binding or activity by the agonist in question. Information Table S3). Phenylephrine and to an even greater Information on candidate receptors can be cross-matched extent methoxamine showed bias towards ERK1/2 with global RNA sequence data or with focused RT-qPCR phosphorylation compared to Ca2+ mobilization (Figure 7B) data. Experimentally, the contribution of candidate receptors and cAMP accumulation (Figure 7C), whereas A61603 canbecheckedbytheuseofsuitableantagonistsand/orby showed bias towards cAMP accumulation compared to confirming a loss of agonist response in cells lacking the ERK1/2 phosphorylation (Figure 7C). receptor. The choice of host cells for GPCR expression is clearly an important consideration. Many groups have used HEK293 fi Discussion cells and we chose these as a 5-HT1B receptor-de cient host cell line. However, like CHO-K1, HEK293 cells endogenously The capacity of drugs to display bias towards different express many known and orphan GPCRs (Table S1). In signalling pathways has important therapeutic implications, untransfected HEK293 cells, noradrenaline produced a both for development of new therapies and for continuing substantial ERK1/2 response, likely associated with α assessment of existing therapies, particularly those with endogenous expression of 2C-adrenoceptors. This was β adverse on-target effects. In an experimental setting, bias or observed previously in HEK293 cells stably expressing 2- functional selectivity has been defined strictly in terms of adrenoceptors, and verified by demonstrating ERK1/2 α conformational bias, that is, the capacity of two drugs to phosphorylation to the 2-adrenoceptor agonist UK14, 304, induce or stabilize distinct receptor conformations displaying and inhibition of the noradrenaline response by the selective measurably different coupling towards effectors for two or antagonist rauwolscine (van der Westhuizen et al., 2014). In more signalling pathways (Kenakin and Miller, 2010). some cases, cell choice overcomes problems associated with β Elegant NMR studies of the 2-adrenoceptor have provided confounding endogenous GPCRs. For example, HEK293 cells direct evidence that biased agonists promote different display robust expression of adenosine A2A and A2B receptor conformations, with Gα-biased ligands favouring receptors (Supporting Information Table S1), but these are movement of TM6 and β-arrestin-biased ligands favouring not present in CHO-K1 cells. In contrast the protease- altered TM7 conformations (Liu et al., 2012). Thus, the activated receptor subtypes PAR1 and PAR2 are expressed in concept and the translational potential of biased agonism both CHO-K1 and HEK293 cells, indicating the need for an are well-founded. On the other hand, it is important to alternative host cell. consider all factors that may contribute to the observation Clinical translation of biased agonism is of critical of apparent bias. We show here that compared to importance, and studies in recombinant cells should be noradrenaline or A61603, oxymetazoline behaved as a followed up by experiments in target cells or isolated tissues super-agonist for ERK1/2 phosphorylation, a partial agonist with direct therapeutic relevance. As described above, the for Ca2+ mobilization, and failed to promote significant μ-receptor agonist TRV130 (DeWire et al., 2013) and the α κ cAMP accumulation in CHO 1A-adrenoceptor cells. However, -receptor agonist triazole 1.1 (Brust et al., 2016) display the profound ERK1/2 phosphorylation response to consistent patterns of bias in HEK293 or CHO-K1 cells oxymetazoline is not related to conformational bias, but expressing receptors recombinantly compared to native cells fl instead re ects off-target effects at 5-HT1B receptors or whole animal experiments. In other cases, however, bias or endogenously expressed in CHO-K1 cells. overall pharmacology may differ markedly depending on the This finding highlights one of the difficulties in experimental system. For example, a comparison of the concluding that agonist behaviour reflects authentic κ-receptor agonists ICI204448 and asimadoline provides a conformational bias. Indeed, there are many issues that can cautionary tale of system effects related to receptor species confound experiments designed to test for biased agonism and the difficult transition from recombinant cell studies that also compromise the translation of bias in recombinant through to animal models and human samples (Broad et al., cells to in vivo therapeutic benefit. We have comprised a 2016). While ICI204448 behaves consistently, studies on checklist of factors that may be important in studies of bias asimadoline reveal substantial differences in efficacy and (Table 2). For example, our study demonstrates strategies for potency between recombinant mouse and human receptors detecting off-target effects, such as the need to check whether expressed in HEK293 cells and native mouse and human all agonists display negligible responses in untransfected colon samples. The authors suggest that asimadoline is a cells, across the entire set of signalling outputs under weak partial agonist, and that this attribute is unmasked only

2328 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP

Table 2 Checklist for the determination of bias

Demonstration of ligand bias Finding Refs Experimental evidence Biased agonism (conformational bias) a,b 1. Bias can be quantiﬁed using the operational model of agonism.

Biased agonists display a ΔΔLog(τ/KA) value significantly different from 0 relative to the reference agonist. 2. In some cases agonists may display a reversal of efficacy or potency across two independent signalling outputs Protean agonism (dual efficacy ligand) c A compound acting as an inverse agonist for one signalling pathway acts as an agonist for a second pathway

Confounding factors Factor Refs Experimental Resolution 1. System bias Off-target activity d 1. Demonstrate lack of response in non-transfected cells, including all signalling outputs with all agonists 2. Demonstrate biased agonism in at least 2 unrelated cell lines Choice of reference agonist e Choice of endogenous agonist as reference may be problematic if it has broad target specificity that includes related receptor subtypes with endogenous expression in host cells Partial agonism c,f Biased agonism must be distinguished from partial agonism. In the latter case apparent bias may reflect signal strength for each assay and not require distinct receptor conformations Level of receptor expression g,h 1. Agonist efficacy is altered in cells with receptor overexpression 2. Effects on observed bias may be skewed depending on signalling assay, therefore test for bias in cells with at least two substantially different receptor expression levels Efficiency of coupling d,e Exercise caution when including signalling outputs with very high coupling efficiency and highly redundant upstream effectors (for example, ERK1/2 phosphorylation) Relevance to cells of therapeutic interest i 1. Use human cell background where possible 2. Follow up observations in therapeutically relevant cells or isolated tissues, and with in vivo experiments 2. Observational bias Kinetic effects j 1. Determine kinetic profiles of biased agonists relative to reference compound 2. Carefully consider choice of time points for each signalling assay in light of kinetic profiles References: (a)(Evanset al., 2011), (b)(Thompsonet al., 2016), (c) (Kenakin and Miller, 2010), (d)currentstudy,(e) (van der Westhuizen et al., 2014), (f) (Luttrell et al., 2015), (g) (Hutchinson et al., 2005), (h)(Satoet al., 2007), (i) (Broad et al., 2016), (j) (Klein Herenbrink et al., 2016)

in the human colon system where there may be a lower reference compound, even though for most receptor families, abundance of κ-receptors, distinct post-translational these agonists display little or no subtype selectivity. This modifications of the C-terminus that reduce effector markedly increases the risk of off-target effects at endogenous coupling, or alternative protein–protein interactions. receptor subtypes, and suggests that more selective Further confounding influences affecting the compounds may be more suitable reference agonists (van identification or quantification of biased agonism are der Westhuizen et al., 2014). A second important summarized in Table 2. In principle, the contribution of cell consideration is that optimization of signalling assay background (system bias) and the particular signalling output measurement time is often done using the reference agonist. being measured (observational bias) should be controlled by This becomes problematic if the kinetics of receptor binding the use of a suitable reference agonist (Evans et al., 2011; by the reference agonist differs appreciably from that of test van der Westhuizen et al., 2014; Klein Herenbrink et al., compounds (Klein Herenbrink et al., 2016). The bias observed 2016). Endogenous agonists are frequently chosen as the under such circumstances may reflect agonist residence time

British Journal of Pharmacology (2017) 174 2318–2333 2329 E D da Silva Junior et al. BJP at the receptor rather than the kinetics of the signalling ERK1/2, depends on individual agonist-GPCR-effector output being measured, as elegantly demonstrated for partial interactions (Halls et al., 2016), and has implications for agonists at the dopamine D2 receptor (Klein Herenbrink et al., cellular events governed by cytosolic phosphorylation 2016). cascades, cytoskeletal changes, or regulation of gene α Oxymetazoline is a weak partial agonist at the 1A- transcription by nuclear signalling. Interestingly, adrenoceptor, raising a third confounding influence on compartmentalized Ca2+ release is also regulated by distinct observations of bias. Partial agonism may be mistakenly signalling pathways. In cardiomyocytes, Golgi Ca2+ release β attributed to conformational bias; however, distinct receptor in response to 1-adrenoceptor stimulation occurs through states are not necessary to explain findings reflecting that a cAMP-Epac/PKA-PLC pathway, distinct from global partial agonists display lower efficacy for signalling outputs sarcoplasmic reticulum Ca2+ release that governs contractile that are poorly coupled to receptor activation (Kenakin function (Yang et al., 2015). Likewise, in HEK293 cells β 2+ and Miller, 2010). This appears to be the case for expressing the 2-adrenoceptor, adrenaline-stimulated Ca α α ε oxymetazoline acting at the 1A-adrenoceptor where cAMP release is dependent on a G s-EPAC-Rap2B-PLC pathway concentration–response curves, even to high efficacy (Schmidt et al., 2001). It is important to note that the effect agonists like noradrenaline and A61603, indicate less of conformational bias on signalling must reflect the efficient coupling compared to Ca2+ release, as potency interaction between GPCRs and the primary effector values are close to 2 orders of magnitude lower (Table 1). protein. Thus if cAMP accumulation and Ca2+ release both One explanation for the failure of oxymetazoline to stimulate result predominantly from coupling of a receptor to Gαs, cAMP accumulation could be the simultaneous activation of there can be no authentic bias though there may be α fi the 5-HT1B receptor to couple to inhibitory G i/o proteins. differences in agonist ef cacy or potency due to signal This was not the case, however, as PTX pretreatment failed strength effects. to unmask an increase in cAMP accumulation to The level of GPCR expression is also a major concern in α oxymetazoline in CHO 1A-adrenoceptor cells (Figure 6C), studies of biased agonism. For example, in CHO-K1 cells with α even though oxymetazoline caused a weak inhibition of the relatively high 1A-adrenoceptor expression, A61603 displays cAMP response in untransfected cells (Figure 6B). However, asignificant 8.6-fold bias towards cAMP accumulation inthepresenceof1μM forskolin, oxymetazoline produced compared to Ca2+ release (Evans et al., 2011), whereas in the α robust cAMP accumulation in CHO 1A-adrenoceptor cells. present study using low-expressing cells, A61603 does not Although the mechanism is not clear, forskolin is known to show significant bias between these two pathways. It could enhance the activation of adenylyl cyclase by hormones be argued that changes in receptor density should alter the or neurotransmitters in other cell systems (Mokhtari et al., ability of all agonists to activate a particular signalling 1988; Paoletta et al., 2014). pathway, but would not change the bias of ligands relative τ Comparison of our cAMP and ERK1/2 data illustrates the to each another or to a reference agonist, since Log( /KA) point about differences in coupling efficiency between values change linearly with receptor density (Kenakin et al., α signalling pathways (Table 2). In CHO 1A-adrenoceptor cells, 2012; Luttrell et al., 2015). This should hold true in systems oxymetazoline couples to the 5-HT1B receptor such that it where the change in receptor density does not alter the ability promotes a substantially greater ERK1/2 response than of the receptor to interact with one or more signalling noradrenaline or A61603, yet at the same time coupling of proteins. However, the potential of GPCRs to couple to α oxymetazoline to the 1A-adrenoceptor overshadows the multiple signalling pathways and the prevalence of an capacity of the 5-HT1B receptor to inhibit cAMP individual pathway may be dependent on the abundance α accumulation in these cells. While 5-HT1B receptor-mediated of receptors. For instance, the ability of G s-coupled ERK1/2 phosphorylation requires Gαi/o coupling and is receptors to stimulate PLC decreases in cells expressing low β β largely blocked by PTX (Figures 2I and 4D), there are levels of 1-or 2-adrenoceptors (Zhu et al., 1994). In βγ β addtional downstream effectors including G and CHO-K1 cells expressing the 3-adrenoceptor, p38 MAPK β-arrestins that may amplify this response (Wurch and responses are influenced by receptor abundance due to an Pauwels, 2000; Wacker et al., 2013). More generally, ERK1/2 interaction between cAMP and p38 MAPK signalling (Sato phosphorylation can be stimulated by receptor coupling to et al., 2007). In cells with higher receptor abundance, Gαs, Gαq/11, Gαi/o and β-arrestins via acomplexnetwork increased cAMP accumulation leads to a reduction in p38 of signalling pathways, and it has been observed that agonist MAPK phosphorylation, resulting in accentuated bias bias is most likely to favour this output (van der Westhuizen between full and partial agonists compared to low- et al., 2014). We also saw preferential bias towards ERK1/2 expressing cells. Therefore, receptor expression levels are phosphorylation over Ca2+ mobilization and cAMP an important factor in determining ligand bias by altering accumulation for phenylephrine and methoxamine in the relative ability of GPCRs to interact with effector α CHO 1A-adrenoceptor cells. proteins, leading to different signalling patterns. This Signalling redundancy could in principle mask authentic phenomenon may complicate the in vivo manifestation of conformational bias, as two agonists may have similar ligand bias as different tissues will display distinct levels of efficacy or potency for ERK1/2 phosphorylation despite expression of signalling proteins and receptors (Kenakin utilizing distinct upstream effectors. This means that a basic and Christopoulos, 2013). definition of effectors and signalling pathways linked to a In conclusion, a valid description of bias requires that the particular response will facilitate the interpretation of biased reference and test agonists all act exclusively at the same agonism. In addition, the cellular location of ERK1/2 receptor subtype, that the measured signalling outputs arise phosphorylation, in particular cytosolic versus nuclear due to direct interaction of the receptor with distinct effector

2330 British Journal of Pharmacology (2017) 174 2318–2333 α Signalling bias at human 1A-adrenoceptors BJP proteins, and that there is no negative interference (or Declaration of transparency and positive crosstalk) between the pathways. Thus, in order to fi properly control for system bias, it is critical that the scienti crigour reference and test agonists act in a parallel manner. This Declaration acknowledges that this paper adheres to the Similarly, observational bias between signalling assays may principles for transparent reporting and scientificrigourof fi be engendered by distinct kinetic pro les of reference and preclinical research recommended by funding agencies, test agonists, requiring appropriate control experiments publishers and other organisations engaged with supporting and careful choice of assay times. We have shown that research. phenylephrine, methoxamine and A61603 display α authentic ligand bias at human 1A-adrenoceptors expressed at relatively low levels in CHO-K1 cells. In contrast, the high efficacy of oxymetazoline for ERK1/2 phosphorylation was attributable to off-target effects at 5-HT1B receptors that are endogenously expressed in these References host cells. Therefore, this study highlights the importance of evaluating ligand bias at a variety of different receptor Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson expression levels and assessing the response selectivity HE et al. (2015a). The concise guide to PHARMACOLOGY 2015/16: G protein-coupled receptors. Br J Pharmacol 172: 5744–5869. (on-target effects) in order to predict the ligand bias in different systems (cells or tissues). Finally, the off-target AlexanderSPH,FabbroD,KellyE,Marrion N, Peters JA, Benson HE effects of oxymetazoline may also have physiological and et al. (2015b). The concise guide to PHARMACOLOGY 2015/16: fi – therapeutic signi cance since 5-HT1B receptors and Enzymes. Br J Pharmacol 172: 6024 6109. α -adrenoceptors are found in the human prostate (Lepor 1A Bauer RM, Strittmatter F, Gratzke C, Gottinger J, Schlenker B, Reich O et al., 1993; Dizeyi et al., 2004; Siddiqui et al., 2006) and et al. (2011). Coupling of alpha1-adrenoceptors to ERK1/2 in the the activation of ERK1/2 phosphorylation in this tissue is human prostate. Urol Int 86: 427–433. known to be related to cell growth and proliferation in prostate cancer or benign prostate hyperplasia (Papatsoris Baycin-Hizal D, Tabb DL, Chaerkady R, Chen L, Lewis NE, Nagarajan et al. and Papavassiliou, 2001; Bauer et al., 2011; Rodriguez- H (2012). Proteomic analysis of Chinese hamster ovary cells. J Proteome Res 11: 5265–5276. Berriguete et al., 2012). Broad J, Maurel D, Kung VW, Hicks GA, Schemann M, Barnes MR et al. (2016). Human native kappa opioid receptor functions not predicted by recombinant receptors: implications for drug design. Sci Acknowledgements Rep 6: 30797. Brust A, Croker DE, Colless B, Ragnarsson L, Andersson A, Jain K et al. We thank Dr. Thomas Chang (Roche Bioscience, Palo Alto, (2016). Conopeptide-derived kappa-opioid agonists (conorphins): α CA) for the gift of plasmid containing the human 1A-4- potent, selective, and metabolic stable dynorphin A mimetics with adrenoceptor cDNA. antinociceptive properties. j Med Chem 59: 2381–2395. EDdaS Jr was supported by National Council for Scientific Campos M, Morais PL, Pupo AS (2003). Effect of cyproterone acetate and Technological Development (CNPq, Portuguese: on alpha1-adrenoceptor subtypes in rat vas deferens. Braz J Med Biol fi Conselho Nacional de Desenvolvimento Cientí co e Res 36: 1571–1581. Tecnológico) of Brazil, Science without Borders Scholarship program (Portuguese: Programa Ciências sem fronteiras), Chen ZJ, Minneman KP (2005). Recent progress in alpha1-adrenergic – process 203518/2014-4. This work was supported by an receptor research. Acta Pharmacol Sin 26: 1281 1287. Australian National Health and Medical Research Council Civantos Calzada B, Aleixandre de Artinano A (2001). Alpha- (NHMRC) program grant (1055134; RJS). DSH was supported adrenoceptor subtypes. Pharmacol Res 44: 195–208. by a NHMRC Career Development Fellowship (545952) and MS by a NHMRC CJ Martin Fellowship (606763). Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471.

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