A dynamic and screening-compatible nanoluciferase-basedcomplementation assay enables profiling of individualGPCR–G protein interactionsReceived for publication, October 26, 2018, and in revised form, December 27, 2018 Published, Papers in Press, December 28, 2018, DOI 10.1074/jbc.RA118.006231

Céline Laschet‡1, Nadine Dupuis‡2, and X Julien Hanson‡§3

From the ‡Laboratory of Molecular Pharmacology, GIGA-Molecular Biology of Diseases, University of Liège, 4000 Liège and the§Laboratory of Medicinal Chemistry, CIRM-Drug Target and Lead Discovery, University of Liège, Liège CHU, B34 (�4),B-4000 Liège, Belgium

Edited by Henrik G. Dohlman

G protein– coupled receptors (GPCRs) are currently the tar-get of more than 30% of the marketed medicines. However,there is an important medical need for ligands with improvedpharmacological activities on validated drug targets. Moreover,most of these ligands remain poorly characterized, notablybecause of a lack of pharmacological tools. Thus, there is animportant demand for innovative assays that can detect anddrive the design of compounds with novel or improved pharma-cological properties. In particular, a functional and screening-compatible GPCR–G protein interaction assay is still unavail-able. Here, we report on a nanoluciferase-based complementationtechnique to detect ligands that promote a GPCR–G proteininteraction. We demonstrate that our system can be used toprofile compounds with regard to the G proteins they activatethrough a given GPCR. Furthermore, we established a proof ofapplicability of screening for distinct G proteins on dopaminereceptor D2 whose differential coupling to G�i/o family mem-bers has been extensively studied. In a D2–G�i1 versus D2–G�o

screening, we retrieved five agonists that are currently beingused in antiparkinsonian medications. We determined that inthis assay, piribedil and pergolide are full agonists for therecruitment of G�i1 but are partial agonists for G�o, that theagonist activity of ropinirole is biased in favor of G�i1 recruit-ment, and that the agonist activity of apomorphine is biased forG�o. We propose that this newly developed assay could be usedto develop molecules that selectively modulate a particular Gprotein pathway.

G protein– coupled receptors (GPCRs)4 are a large family ofmembrane proteins that have pivotal functions in physiology

and are directly targeted by more than 30% of our therapeuticarsenal (1). Given their successes in drug discovery, it is com-mon sense to postulate that uncharacterized members holdgreat potential in terms of innovative therapeutic strategies(2–5). Several authors recently proposed that the paucity ofadequate pharmacological tools was precluding research onelusive receptors (5, 6). In addition, there is still an unmet med-ical need in various diseases for drugs with improved propertiessuch as increased potency, higher selectivity, or refined efficacyfor existing validated drug targets. Thus, the drug discoveryprocess would benefit from more sophisticated assays able todetect with maximal accuracy the ligands with a desired phar-macological profile.

GPCR signaling has been extensively studied and is notori-ously complex. The current paradigm states that the binding ofa ligand to its receptor stabilizes active conformations that inturn triggers through allosteric effects the formation of anactive complex bound with intracellular partners (7). It is gen-erally accepted that the prime event following ligand binding isthe interaction of the active receptor with heterotrimeric Gproteins composed of � and �� subunits (8). These elementsdissociate upon activation, and each of them has the capacity topromote distinct signaling pathways (9). Following G proteinactivation, the receptor undergoes desensitization and inter-nalization through diverse processes involving phosphoryla-tion by GPCR-specific kinases (GRK) and scaffolding by pro-teins such as arrestins (10).

In humans, the G protein family comprises 16 members thatare classified according to the identity of their � subunits intofour families (11). The G�s family (G�slong, G�sshort, and G�olf)increases and the G�i/o family (G�i1, G�i2, G�i3, G�o, and G�z)decreases adenylate cyclase activity. Hence, G�s/olf and G�i/ooppositely regulate levels of cAMP in the cell (9). The G�q/11family (G�q, G�11, G�15, and G�16) notably triggers the activa-tion of the phospholipase C-� and calcium mobilizationthrough the release of diacylglycerol and inositol triphosphate(9). G�12 and G�13 (G�12/13) activate the small GTPase regula-tors Rho-GEF (9). Although individual receptors were initially

This work was supported by the Fonds pour la Recherche Scientifique (F.R.S.-FNRS) Incentive Grant F.4510.14 for Scientific Research, University of Liège(Fonds Spéciaux), and the Léon Fredericq Foundation. The authors declarethat they have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S5.1 F.R.S.-FNRS Ph.D. fellow.2 FRIA Ph.D. fellow.3 F.R.S.-FNRS research associate. To whom correspondence should be

addressed: Laboratory of Molecular Pharmacology, GIGA, University ofLiège, CHU, B34 (�4), 11 Ave. de l’Hôpital, B-4000 Liège, Belgium. Tel.:32-4-3664748; Fax: 32-4-3664198; E-mail: j.hanson@uliege.be.

4 The abbreviations used are: GPCR, G protein– coupled receptors; GRK, GPCRkinase; D2, long isoform of the dopamine receptor 2; NanoBiT, Nano-Luciferase Binary Technology; BRET, bioluminescence resonance energytransfer; FRET, fluorescence resonance energy transfer; LgB, large BiT; H3,

histamine receptor 3; SUCNR1, succinate receptor; NP, natural peptide;NanoLuc, nanoluciferase; �2AR, �2-adrenergic receptor; H1, histaminereceptor 1; TP�, thromboxane A2 receptor �; RLU, relative luminescenceunit; RET, resonance energy transfer; PD, Parkinson’s disease; GTP�S,guanosine 5�-3-O-(thio)triphosphate; AA, amino acid.

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seen as being selective for a given pathway and thus coupled toa single G protein, it has been observed that most receptorsdisplay at least some level of promiscuousness toward differentG proteins or G protein families (9). Another layer of complex-ity exists in GPCR signaling with the observation of functionalselectivity, which can be defined as the ability of a ligand tostabilize a specific receptor conformation leading to a uniqueand ligand-determined profile of signaling pathway activations(7).

A large array of pharmacological assays has been developedfor the identification of substances able to modulate G proteinsthrough GPCRs. Most of them focus on the downstream eventstriggered by G protein activation such as the generation ofcAMP (G�s/olf and G�i/o) (12, 13), Ca2� (14) and inositolmonophosphate (G�q/11), the downstream activation of genepromoters, or more recently, the shedding of transforminggrowth factor � (G�q/11 and G�12/13) (15). More generic andholistic assays measuring the accumulation of the nonhydroly-sable GTP�S (reflecting G protein activation) (16) or cell mor-phology (17) have also been extensively used. To extend thescope of an assay to more than one or two pathways, promiscu-ous G proteins linking most receptors to common second mes-sengers are generally added to the system (18). Collectively,these assays are limited in the way that they cannot give directinformation on the kind of G protein, or even on the identity ofthe GPCR, that has been activated by a given ligand. Thus, dur-ing screening campaigns, they are prone to deliver a high rate offalse positives (13).

More recent techniques based on resonance energy transfer(BRET and FRET) have given unprecedented access to thestudy of individual interaction between G protein and theirreceptors in living cells (19). However, these approaches sufferfrom important drawbacks such as limited sensitivity (BRET)or high background noise (FRET) that limit their use, forinstance in high-throughput screenings (20). In addition, theyrequire the presence of bulky donor and/or acceptors, although

reduced-size donors such as NanoLuc (BRET (21)) or FlasH(FRET (22)) are now routinely used.

Here, we describe a simple and flexible NanoLuc-based com-plementation assay that overcomes these issues and givesaccess to the profiling of ligands for their ability to induceGPCR interaction with individual G protein. In addition, theprocedure is compatible with the settings of high-throughputscreening. We applied this methodology to several prototypicalclass A receptors and their cognate G proteins. The D2 receptoris a well-validated drug target in psychosis and Parkinson’s dis-ease and is coupled to the G�i/o family. We used the D2 receptoras a proof-of-concept to perform a screening of a library con-taining known drugs and active compounds against the D2receptor.

Results

NanoLuc complementation can monitor receptor–G proteininteraction in living cells

To detect the real-time interaction between receptor and Gproteins, we selected the NanoLuc Binary Technology (Nano-BiT) that is based on NanoLuc, an engineered luciferase fromthe deep sea shrimp Oplophorus gracilirostris (23). We rea-soned that the reported increased brightness of the enzymewould overcome sensitivity issues of other systems, such as fire-fly luciferase complementation, BRET or FRET. In addition, weexpected the small size of the NanoBiT partners (a small sub-unit (SmBiT, 11 AA)) and a larger subunit ((LgBiT, 158 AA),Fig. 1A (24)) could minimize perturbations of GPCR pharma-cology that have been outlined with large fluorescent proteins(19).

As proof of concept for such an approach, we first selectedthree receptors coupled to the G�i/o family: the long isoform ofthe dopamine receptor subtype 2 (D2) (25); the histaminereceptor H3 (26); and the succinate receptor (SUCNR1) (27).The SmBiT was attached to the C terminus of the receptors,

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Figure 1. NanoLuc complementation system. A, constructs for the NanoLuc complementation assay between a GPCR linked to SmBiT, NP, or HiBiT peptidesand a G� subunit linked to LgBiT. Upon stimulation (yellow circle), the coupling of heterotrimeric G protein to the receptor triggers the exchange of a GDP(purple star) by a GTP (green star) followed by dissociation of G� from G�� subunits. The interaction between GPCR and G� subunit induces the formation ofa complete NanoLuciferase and light emission in the presence of its substrate. B, amino acid sequence and KD value of the different small peptides (SmBiT, NP,and HiBiT). Red amino acids represent mutated amino acids compared to NP.

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and the LgBiT was introduced in the loop connecting helices Aand B of the G�i1 protein (G�i1—LgB91) (Fig. 1A). The interac-tion between labeled receptor and G�i1–LgB91 was estimatedby measuring the emitted light upon agonist stimulation. Allthe tested receptors induced a rapid increase of luminescentsignal upon stimulation (Fig. 2, A–C). However, the amplitudeand stability of the obtained signal was relatively weak. We rea-soned that the transient nature of the GPCR–G protein inter-action could, to some extent, explain the low level of theobserved signal. SmBiT has been optimized to have a low affin-ity for LgBiT (KD � 190 �M (24)) compared with the nativesequence (natural peptide or NP, KD � 0.9 �M (24)) to minimizethe perturbation of the physiological interaction induced by thepresence of complementing partners (24). A third peptide withhigh affinity (HiBiT, KD � 0.7 nM (24)) has also been described(Fig. 1B). To improve the signal-to-noise ratio of our assay, wehypothesized that the affinity of the small complementing pep-tide could be a critical parameter for the sensitivity of the detec-tion system. Therefore, we tested the other two peptides, NPand HiBiT, fused at the same location as SmBiT on the testedreceptors. For the three receptors, the amplitude and the sta-bility of the signal were markedly increased when NP was used

as the complementation peptide (Fig. 2, D–F). For both thesepeptides, the signal remained stable for at least 10 min (Fig. 2,D–I), which is consistent with the literature describing real-time GPCR–G protein interaction (28, 29).

NanoLuc complementation is completely reversible whennatural peptide or SmBiT are fused to the receptor

Next, we envisaged the possibility that the use of comple-menting partners with different affinities for each other couldcompletely distort the GPCR–G protein interaction andincrease the risk of detecting nonpharmacological interactionsand the accumulation of irreversible receptor–G protein com-plexes. Thus, we tested the reversibility of the complementa-tion between the three small peptides (SmBiT, NP, and HiBiT)and LgBiT in our system. Following stimulation with dop-amine, a competitive D2 receptor antagonist (sulpiride) wasinjected. For the D2 receptor fused with the SmBiT, a rapiddecrease of the NanoLuc signal that reached basal level wasobserved upon antagonist addition (Fig. 3A). When the exper-iment was repeated with the constructs containing NP, a simi-lar pattern was observed, although the signal-to-noise ratio wasincreased (Fig. 3B). When the HiBiT was used as a partner, the

Figure 2. NanoLuc complementation measurement of interaction between G�i1 and GPCRs linked to SmBiT, NP, or HiBiT peptides in living cells.NanoLuc activity kinetics measured in HEK293 cells co-expressing G�i1–LgB91 and GPCRs linked to three different small peptides are indicated in each panel.A–C, D2, H3, and SUCNR1 linked to SmBiT; D–F, D2, H3, and SUCNR1 linked to NP; G–I, D2, H3, and SUCNR1 linked to HiBiT, before and after injection of theirrespective ligand (dopamine 1 �M, imetit 100 nM, and succinate 1 mM). Results are expressed as the normalization of the NanoLuc signal in the presence ofagonist to the signal in the absence of agonist. Data are representative of the mean � S.E. of at least three independent experiments.

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rate of dissociation was markedly decreased, and the signal didnot reach the basal level, suggesting an incomplete dissociationof the receptor–G protein complex (Fig. 3C). In light of suchresults, we decided to select the system using the NP for furtherinvestigations.

GPCR–G protein NanoLuc complementation can be applied toall G protein subtypes

We questioned the possibility of implementing the detectionsystem to other G protein subtypes. Thus, we also expanded our

detection system to other families of G� proteins. We intro-duced the LgBiT in the loop connecting helices A and B of theother G� proteins and tested the long isoform of G�s with the�2-adrenergic receptor (�2AR) (Fig. 4A), G�q and G�11 withthe histamine receptor H1 (H1) (Fig. 4B), and G�12 and G�13with the thromboxane A2 receptor (TP�) (Fig. 4C). Upon stim-ulation with their respective ligands, a significant interactionbetween the receptor and the G� subunit was recorded in cellsco-transfected with �2AR-NP/G�s–LgB113, H1-NP/G�q/11–LgB97, and TP�-NP/G�12/13–LgB115/106 (Fig. 4, A–C). We thenaimed at performing a complete profiling of G protein recruit-ment on a test receptor. We chose the well-characterized D2dopaminergic receptor as a model because it has been previ-ously reported to couple differently to a diversity of G proteinsfrom the G�i/o family (30). We designed additional sensors forthe other � subunits of the Gi/o family. We inserted the LgBiT atthe same topological location in G�i2 and G�i3. For G�o (the “a”isoform), the initial constructs gave a poor signal, and afterdifferent rounds of optimization, the LgBiT was placed justafter helix C, and a deletion of the first 52 amino acids at theN-terminal side (G�o–LgB143d) was performed. We stimulatedwith dopamine the HEK293 cells transiently transfected withD2–NP and each of these G� subunits fused with LgBit. Expo-sure of D2–NP to its endogenous agonist, dopamine, induced asignificant increase of luminescence signal when the receptorwas co-expressed in HEK293 cells with either G�i1–LgB91,G�i2–LgB91, G�i3–LgB91, or G�o–LgB143d but no activity whenthe D2–NP was co-transfected with plasmids containing thevalidated constructs G�s–LgB113, G�q–LgB97, G�11–LgB97,G�12–LgB115, or G�13–LgB106 (Fig. 4D). The interaction pro-file of the D2 with G� subunits that was obtained with the pres-ent system was consistent with the ones already described in theliterature (31–33).

GPCR–G protein NanoLuc complementation system isamenable to high-throughput screening

We further examined whether the GPCR–G protein Nano-Luc complementation assay could be used for a high-through-put screening campaign. To transpose this assay to a screeningprotocol, we first optimized the G�i1 construct. The LgBiT wasinserted at different locations of the G� protein and tested inthe presence of D2 receptor tagged with the NP (Fig. S1A). TheLgBiT placed at the N terminus of G�i1 (G�i1–LgBN-term)showed the strongest signal among all the constructs tested.Similar investigations on G�o confirmed that G�o–LgB143d wasthe construct that gave the best signal-to-noise ratio (Fig. S1B).Next, we performed a flow cytometry experiment to determinethe relative expression of the D2–NP receptor at the membranecompared with the intracellular expression of G�i1–LgBN-term

and G�o–LgB143d in transiently transfected HEK293 cells. Weobserved a similar expression of receptor and G� protein con-structs for the two conditions of transfection (Fig. S2). Toobtain the higher signal-to-noise ratio, we tested a differentstoichiometry of D2–NP and G�i1–LgBN-term/G�o–LgB143d forthe transfection. A ratio 1:1 of receptor and G� subunitrevealed a higher signal-to-noise ratio and was used for thesubsequent experiments (Fig. S3A). Next, we reasoned that theG�� dimer could affect the amplitude of the signal obtained, as

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Figure 3. Reversibility of the SmBiT, NP, and HiBiT peptides. NanoLucactivity was measured in HEK293 cells co-expressing G�i1–LgB91 and D2linked to SmBiT (A), NP (B), or HiBiT (C), before and after stimulation with thereceptor ligand dopamine (1 �M). Twenty minutes after addition of agonist, aselective-receptor antagonist sulpiride (10 �M) was injected. Data representstimulated cells normalized to nonstimulated cells. Data are representative ofthe mean � S.E. of at least three independent experiments.

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it has been shown for the RET-based systems (28). However, theco-expression of G�o–LgB143d with its complementary G�1�2dimer did not modify the recruitment of G�o–LgB143d toD2–NP. Interestingly, the co-expression of D2–NP and G�i1–LgBN-term with G�1�2 subunits decreased the signal of Nano-Luc complementation upon dopamine stimulation, although itdid not affect the pEC50 (Fig. S3B). Thus, the detection of theinteraction between NP-tagged GPCR and the LgBiT-taggedG� subunit does not require the co-expression with the G��dimer.

Concentration-response curves that were determined on thesystem revealed specific interaction between D2–NP/G�i1–LgBN-term (EC50 � 2 nM) and D2–NP/G�o–LgB143d (EC50 �116 nM) in the presence of dopamine (Fig. 5, A and B). Duringthe kinetic measurement of the D2–NP/G�i1–LgBN-term or theD2–NP/G�o–LgB143d interaction induced by dopamine stimu-lation, we observed an increase of signal that started to decreaseimmediately upon competitive antagonist (sulpiride and spip-erone) addition (Fig. 5, C and D). The affinity of sulpiride for theD2–NP construct that was estimated with a Schild plot (Fig. S4)was not significantly different between the two assays and wasconsistent with the literature (34). We applied statistical meth-ods to determine whether this assay would be suitable for high-throughput screening. We calculated the Z� factor, whichincludes the mean and the standard deviation of the positiveand negative control (35). This factor reflects the dynamicrange of the assay and its ability to detect active ligands. A Z�factor that is between 0.5 and 1 is characterized by a large bandseparation between positive and negative signals and can beconsidered as an excellent assay for hit identification (35).When cells co-expressing D2–NP with G�i1–LgBN-term orG�o–LgB143d were stimulated with either dopamine or thevehicle in a 96-well-plate, the Z� factors were of 0.61 and 0.69,respectively (Fig. 5, E and F).

G�i1- and G�o-based screening of a SOSA library identifies D2

agonists with distinct pharmacological profiles

To further validate our approach, we screened a SOSAlibrary composed of 1200 known active compounds and drugs(Prestwick Chemical Library�) on D2–NP-expressing cellstogether with the G�i1- or G�o-based complementation con-

structs (G�i1–LgBN-term or G�o–LgB143d) in parallel. Severalagonists of the D2 receptor were present in this library, and wereasoned that they would serve as internal positive controls. Wefixed the threshold for hit identification as the mean of thenegative control plus three times the standard deviation. Withthis criterion, we detected the six agonists (the D2 agonists werelisted according to the BJP/IUPHAR database http://www.guidetopharmacology.org)5 (53) of the D2 receptor present inthe library (Fig. 6, A and B).

Next, we tested a wide range of agonist concentrations andcompared the profile of each ligand with regard to dopaminethat was defined as the reference ligand. Surprisingly, the pEC50of dopamine was very different between the G�i1- and G�o-based interaction assay (Table 1; pEC50: 8.64 � 0.10 and 6.60 �0.05, respectively). We postulated that the difference betweenpEC50 could be the consequence of using different constructs.Actually, when we tested several constructs of G�i1–LgB, wealso obtained a difference of pEC50 upon stimulation with dop-amine (Fig. S1).

We compared all data obtained with both G�i1 and G�oassays relative to dopamine. We observed that the Emax of theD2 agonists were similar when we measured the interactionbetween D2–NP and G�i1–LgBN-term (Table 1). However, whencells expressing D2–NP and G�o–LgB143d were stimulated withapomorphine, piribedil, and pramipexole, we detected lowerefficacies compared with the one of dopamine as these com-pounds behaved as a partial agonist for the initiation of inter-action between D2 and G�o. For example, piribedil exhibited adecrease of efficacy up to 3-fold compared with the one mea-sured for dopamine (Fig. 7, A and B). Furthermore, althoughpergolide was more potent than dopamine as an inducer of boththe D2–NP/G�i1–LgBN-term and D2–NP/G�o–LgB143d interac-tions (Table 1; pEC50 of 10.13 � 0.07 and 8.26 � 0.07 for G�i1–LgBN-term and G�o–LgB143d, respectively), we observed a lowerefficacy for pergolide when measuring the interaction betweenD2–NP and G�o–LgB143d. Ropinirole-promoted D2–NP/G�i1–LgBN-term and D2–NP/G�o–LgB143d interactions wereboth less potent compared with dopamine (Fig. 7, A and B). In

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Figure 4. NanoLuc complementation measurement of GPCR and G protein interactions in living cells. A–D, NanoLuc activity was measured in HEK293cells co-expressing G� subunit linked to LgBiT and GPCR linked to NP as indicated in each panel. Cells were stimulated with vehicle or with their respectiveagonist (isoproterenol, histamine, and dopamine 10 �M; U46619 10 nM), and the results are expressed as the normalization of agonist-treated cells to untreatedcells. Data represent the mean � S.D. of three independent experiments performed in triplicate. Statistical significance between stimulated and unstimulatedcells was assessed using a nonparametric Mann-Whitney test (***, p � 0.001).

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addition, relative to dopamine, ropinirole showed a higherpotency for inducing the D2–NP/G�i1–LgBN-term comparedwith the D2–NP/G�o–LgB143d interaction (Table 1; pEC50 of8.23 � 0.06 and 5.89 � 0.08 for G�i1 and G�o, respectively).

For the different tested ligands, there were no marked differ-ences (when compared with dopamine) between the pEC50 for

D2–NP and G�i1–LgBN-term or D2–NP and G�o–LgB143d inter-actions (Fig. 7C). However, when we considered the Emax, par-tial agonism was recorded when measuring the interactionbetween D2–NP and G�o–LgB143d (Fig. 7D). This difference inmeasured Emax was more notable for piribedil and pergolide(Fig. 7D). For each tested ligand, �log(Emax/EC50) was

Figure 5. Screening compatible NanoLuc complementation assay for GPCR and G protein interactions. HEK293 cells co-expressing D2–NP/G�i1–LgBN-term or G�o–LgB143d are shown. A and B, concentration-response curves of dopamine. C and D, kinetic measurements of NanoLuc activity duringstimulation with dopamine and after addition of two different antagonists (sulpiride and spiperone). E and F, assay performance (Z� factor determination) forD2 untreated and treated with dopamine (10 �M) on transiently co-transfected cells (n � 48). Data are representative of the mean � S.E. of at least threeindependent experiments.

Figure 6. Screening of Prestwick Chemical Library� on D2–G�i1 and D2–G�o. A and B, 1200 compounds were tested at the same concentration (5 �M) oncells co-expressing D2–NP/G�i1–LgBN-term or G�o–LgB143d. Results are expressed as the ratio over vehicle (DMSO)-treated (compounds) cells.

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determined for both G� constructs using dopamine as thereference ligand, followed by the calculation of the bias fac-tor as ��log(Emax/EC50) � �log(Emax/EC50)G�i1 pathway ��log(Emax/EC50)G�o pathway according to a previously de-scribed method (36). A bias factor of 0 corresponds to absenceof bias (relative to the reference ligand, dopamine), whereas abias factor of 1 would mean a 10-fold preference for inducingthe G�i1 interaction. On the contrary, a negative value wouldmean a preference for the G�o-dependent pathway. A bias fac-tor of �0.27 (�0.03) (apomorphine), �0.12 (�0.18) (piribedil),0.04 (�0.17) (pramipexole), 0.41 (�0.12) (ropinirole), and 0.09(�0.18) (pergolide) was computed for D2 agonists, demonstrat-ing a significant bias for ropinirole and apomorphine throughG�i1–LgBN-term and G�o–LgB143d, respectively (Fig. 7E).Thus, the GPCR–G protein NanoLuc complementation assayis able to identify biased agonists for certain G proteins.

Furthermore, we tested the possibility of an extension of thisassay to detect �-arrestin recruitment. Thus, we also evaluatedthese five D2 agonists using dopamine as a reference ligand ina �-arrestin 2– based NanoLuc complementation assay. Weobserved a partial activity for apomorphine, piribedil, rop-inirole, and pergolide, whereas pramipexole was a full ago-nist for �-arrestin 2 recruitment (Fig. S5). This assay wasbased on the same D2–NP construct and a �-arrestin 2tagged with the LgBiT at the N terminus. Thereby, we wereable to monitor G protein or �-arrestin interaction using thesame readout.

Discussion

In this study, our first goal was to develop a simple, robust,and sensitive GPCR assay for the real-time detection ofreceptor–G protein interaction that would be amenable tohigh-throughput screening.

The protein-fragment complementation assays have beendevised on the principle that two complementary parts of areporter protein fused to two putative partners would bedetectable upon interaction (37). These strategies have greatlyadvanced our understanding of cellular biology, molecular biol-ogy, or pharmacology. However, although they have a relativelyhigh signal-to-noise ratio, they have been so far less popularcompared with RET to monitor GPCR–G protein interactions.The main reason for this is that the complementation may per-turb the natural interaction between the partners under scru-tiny because of a possible intrinsic affinity between the two splitparts of the reporter proteins or the irreversibility of the com-

plementation once formed. For example, systems based on thefluorescent protein of the GFP family or some �-galactosidasesare unable to dissociate once re-formed and to accumulate inthe system (38). The firefly and Gaussia luciferases seem tohave a reduced propensity to form stable complexes, but thesplit parts have affinity for each other, and once reformed theyare not very bright (39, 40).

Recently, a novel protein fragment complementation basedon a brighter and smaller luciferase called NanoLuc has beendescribed (24) and was already applied in the GPCR field todetect receptor–arrestin association (24, 39, 41). Nano-Luc-based complementation presents several advantages com-pared with other RET- or complementation-based techniques.First of all, the system does not suffer from sensitivity issuesbecause the protein that detects the interaction is a brighterluciferase (42). A second improvement is the small size of boththe reconstituted luciferase and one of the complementingpartners (13 AA only for the SmBiT, 11 AA for NP) that mini-mizes the risk for artifacts that would be induced by the bulkynature of the system constituents (24). A third advantage thatwe noticed is the dynamic reversibility of the system, at leastwhen the SmBiT and NP were used on H3, D2, and SUCNR1(see Figs. 3B and 5, C and D). It is important to note that wedid not formally demonstrate that the true dynamic of theGPCR–G protein interaction was preserved. Therefore, wecannot exclude that the rate of association/dissociation isimpaired as a consequence of the complementation. Notwith-standing, the basal affinity of the complementing partnerscould still promote artifactual constitutive activity by bringingthe G protein and the receptor in close vicinity. However, thisassay was not designed to study these dynamics but to detectactive ligands for uncharacterized receptors. Thus, the possibleincrease of sensitivity may actually be seen as beneficial.

Several strategies have been successfully applied in the pastto specifically monitor the G protein that interacted with or wasactivated by a receptor bound by an agonist. Historically, thefirst reported attempts used cellular backgrounds such as theyeast or insect cells that are devoid of most of the G proteins orGPCR present in human cell lines such as HEK293 (43).Another screening-compatible assay that is able to monitor theactivation of each G protein individually has been describedrecently (33). This elegant approach detects the interaction ofthe activated G protein (G�� subunits) with GRK3 with aNanoBRET system and was applied to profile the couplingbetween receptors and a large set of G proteins (33). Ourapproach differs in several aspects compared with the onedescribed by Masuho et al. (33). First, some G proteins, suchas G�12, were not detectable by their system (33). Second,although the sensitivity and signal-to-noise ratio is relativelyhigh, the authors did not test the possibility of using that assayfor a screening campaign. Third, the nature of the activatingreceptor giving rise to the recorded signal is not identifiable in asystem solely based on G protein activation. Thus, a screeningcampaign on an elusive receptor with the assay described byMasuho et al. (33) would give a high rate of false-positiveligands that are activating the endogenous GPCRs present onthe cell surface. Another putative way to monitor single G pro-tein interaction to a receptor was published during the prepa-

Table 1Agonist activity on G�i1 and G�o interaction of D2 ligandsAgonist-induced G�i1 or G�o interaction with D2 is shown. Efficacy (Emax) is relativeto the maximum effect of dopamine (�100%). Bias factor was calculated asdescribed under “Experimental procedures.” Values in parentheses indicate S.E.

LigandGi1 Go Bias factor, ��log

(Emax/EC50)pEC50 Emax pEC50 Emax

% %Dopamine 8.64 (0.10) 100 6.60 (0.05) 100 0 (0.12)Apomorphine 8.25 (0.04) 103 6.62 (0.04) 63 �0.27 (0.03)Piribedil 8.28 (0.04) 112 6.76 (0.14) 42 �0.12 (0.18)Pramipexole 8.59 (0.07) 119 6.80 (0.09) 77 0.04 (0.17)Ropinirole 8.23 (0.06) 106 5.89 (0.08) 73 0.41 (0.12)Pergolide 10.13 (0.07) 100 8.26 (0.07) 54 0.09 (0.18)

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ration of this manuscript. Using G proteins truncated at theirN-terminal parts that were initially developed to facilitate crys-tallographic studies (called mini G protein), Wan et al. (44)reported that they could monitor G protein–GPCR interac-tions but did not challenge the assay in screening conditions.Interestingly, we applied independently a reduction of the sizeof the G protein to optimize our G�o construct, further validat-ing this strategy to improve the performance of engineered Gproteins. Actually, all the constructs we developed did not givethe same results in terms of sensitivity or signal-to-noise ratio.It should be taken into account when implementing the systemfor other receptors that several constructs should be tested toidentify the best ones. This aspect is of particular importancefor orphan receptors because the confidence in assay perfor-mance is critical if no positive control exists.

BRET and FRET approaches have been extensively used tomonitor G protein recruitment. In principle, a donor (a lucifer-ase in the case of BRET or fluorescent protein in the case ofFRET) is fused to one of the interacting partners, and an accep-tor is fused to another. In case of sufficient proximity (the donorand acceptor must be at a distance below 10 Å with a correctorientation (19)), the fluorescence of the acceptor can bedetected at a unique wavelength, whereas the recorded emis-sion of the donor will be reduced (19). Although these elegantRET approaches shed light on exquisite aspects of receptors,pharmacology such as G protein–GPCR association/dissocia-tion rate or G protein recruitment fingerprints for individualligands (29, 45, 46). They were never applied to library screen-ings probably because of their limited sensitivity. In addition, itshould be noted that for RET systems to work, the presence of

G�� is required with precise stoichiometry, which further limittheir broad use, especially for screening campaigns. Here,we have demonstrated that the NanoLuc complementationapplied to G� does not necessitate the co-transfection of addi-tional G�� subunits (Fig. S3B). For the ease of comparison, wehave listed in Table 2 the advantages and limitations of differentmethods available to monitor G protein–GPCR interactions inliving cells.

The dopaminergic system is composed of five dopaminereceptors (D1 and D5 principally coupled to G�s/olf and D2-4coupled mainly to G�i/o family) of which several are validatedtherapeutic targets for debilitating conditions such as Parkin-son’s disease (PD) or psychotic disorders (30). PD is a degener-ative disorder marked by tremor, rigidity, and slowness ofmovement due to a decline of dopaminergic neurons in thesubstantia nigra (47). In general, the dopaminergic drugs usedto treat PD aim at restoring dopamine signaling in affectedareas such as the striatum, notably through the activation of thereceptor from the D2 family (48). The screening of a library ofknown drugs on the D2 receptor with a novel GPCR–G proteininteraction assay detected five D2 receptor agonists that arecurrently in use to treat patients suffering from PD (49). Thefull concentration-response curves that we determined forD2–G�i1 and -G�o interactions showed that although therewere no relative differences (compared with dopamine) withregard to the potencies of the agonists (except for ropinirole),their Emax displayed signs of partial agonism when recruitingG�o, in particular for piribedil and pergolide (see Fig. 7 andTable 1). The bias factors that we calculated indicate a differentpharmacological profile for the different compounds, especially

Figure 7. Pharmacological characterization of D2 agonists on G�i1 and G�o coupling. A and B, concentration-response curves of five D2 agonists on G�i1and G�o interaction. Emax is expressed as the percentage of dopamine maximal activity. Estimation of pEC50 values (�S.D.) for each ligand and agonist efficacymeasurements relative to dopamine are provided in Table 1. Correlation between the pEC50 (�S.D.) (C) and Emax (�S.D.) (D) values determined in G�i1 and G�oassays. E, bias factors (��log(Emax/EC50)) (�S.D.) calculated for tested D2 agonists; positive values indicate bias for G�i1- over G�o-dependent pathway, whereasnegative values indicate bias for G�o-dependent pathway. Unpaired Student’s t tests were performed on the bias factors to determine the significance ofligand biases between G�i1 and G�o pathways (*, p � 0.05). Data points are representative of at least three independent experiments performed in triplicate.

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ropinirole and apomorphine. At this stage, the demonstrationthat the partial agonism on G�o is linked to the therapeuticeffect would be premature, but the assay presented here has thepotential to facilitate the discovery of compounds with a morepronounced bias that could be evaluated for a beneficial thera-peutic effect.

Furthermore, the differences in the pharmacological profilewe observed between G�i1 and G�o with the NanoLuc comple-mentation on the D2 agonist are consistent with the literature.Pioneering work by Strange and co-workers (32, 50) in insectcells demonstrated that different agonists had the ability toelicit different responses depending on the G protein subtype.Using G protein mutants resistant to pertussis toxin treatment,Milligan and co-workers (31) managed to show similar behav-ior of the D2 receptor toward different G proteins of the G�i/ofamily.

The detection of biased agonism is currently based on thedifferences between activation of G proteins- versus arrestins-based signaling. Usually, the assays to monitor G protein cou-pling rely on second messengers generation while the arrestinrecruitment is estimated with a complementation assay (51).However, we think that it would be preferable to estimate biaswith a common strategy instead of different assays because theassays are an important source of artifacts when determiningbias. We demonstrated here that the nanoluciferase comple-mentation could also be applied to the estimation of the arrestinrecruitment (Fig. S5). Thus, a NanoLuc complementation canbe applied to a more robust analysis of biased agonism betweenarrestin (or any other intracellular partner, in theory) and agiven G protein.

In conclusion, this study describes the development and use-fulness of a novel system for the detection of direct interactionsbetween GPCR and single G protein. This assay has a dynamicrange that is compatible with high-throughput screening. Itopens new avenues for programs aimed at the identification inlarge libraries and optimization of original scaffolds character-ized by biased agonism at the level of G protein subtype, a fea-ture that would be impractical with current technologies.Exquisite pharmacological tools such as biased agonists selec-tively promoting the interaction of a receptor with a restrictedset of G proteins should ease our understanding of the physio-logical rationale for multiple coupling and apparently redun-dant G proteins.

Experimental procedures

Materials

Dopamine, isoproterenol, succinate, histamine, imetit,sulpiride, piribedil, pramipexole, and pergolide were fromSigma. Ropinirole and U46619 were from Santa Cruz Biotech-nology (Dallas, TX); spiperone was from Abcam (Cambridge,UK). Prestwick Chemical Library� was from Prestwick Chem-ical (Illkirch, France).

Plasmids

Human D2 long, G�i2, and G�i3 were amplified from humanORFeome (version 7.1, http://horfdb.dfci.harvard.edu/hv7/).5Human H1, H3, TP�, G�oa, and G�s were amplified frompcDNA3.1� coding for each protein (cDNA Resource Center,T

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Bloomsburg, PA). The SUCNR1-coding sequence was ampli-fied from genomic DNA of human embryonic kidney 293(HEK293) cells. �2AR, G�i1, G�q, G�11, G�12, and G�13 wereamplified from the cDNA of HEK293 cell mRNA. All receptorswere cloned into the pcDNA3.1� (Invitrogen) after addition ofthe FLAG epitope (DYKDDDDK) at the N terminus, precededby the signal sequence KTIIALSYIFCLVFA (54) for D2 and�2AR. After cloning into the pcDNA3.1�, the SmBiT (VTGYR-LFEEIL), NP (GVTGWRLCERILA), and HiBiT (VSGWRLFK-KIS) were added with a flexible linker (GNSGSSGGGGSGG-GGSSG) in-frame to the C terminus of the receptor by poly-merase chain reaction (PCR). All G proteins were cloned intothe pcDNA3.1� (Invitrogen) after addition of the HA (YPYD-VPDYA) epitope at the N terminus. Then, EcoRV and SacIIsites (except BamHI and SacII for G�12) were inserted by PCRbetween specific amino acid residues of the G� protein to insertthe LgBiT. The coding sequence of LgBiT (VFTLEDFV-GDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIV-RSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDD-HHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKI-TVTGTLWNGNKIIDERLITPDGSMLFRVTINS) was ampli-fied by PCR and inserted, flanked by a flexible linker (SGGGGS)and the respective restriction sites, into the G� subunitsequence. The location of the LgBiT was topologically identicalfor each of the following G� subunits: between residues 91 and92 of G�i (G�i1–LgB91, G�i2–LgB91, and G�i3–LgB91) andG�oa (G�o–LgB91); residues 97 and 98 of G�q/11 (G�q–LgB97

and G�11–LgB97); residues 113 and 114 of G�s (G�s–LgB113);residues 115 and 116 of G�12 (G�12–LgB115); or residues 106and 107 of G�13 (G�13–LgB106). The G�i1 constructs used forthe screening have been obtained as follows: the LgBiT wasadded at the N terminus of G�i1 by cloning its coding sequenceinto the pNBe3 vector (Promega Corp., Madison, WI) withXhoI and SacI sites, and a HA tag was added at the C-terminalsequence by PCR (G�i1–LgBN-term). The G�oa construct usedfor the screening was obtained by insertion of the LgBiT andflanked by a flexible linker (SGGGGS) and EcoRV/SacII sites,between residues 143 and 144 of G�oa with a HA tag at the Nterminus cloned into the pcDNA3.1� (Invitrogen). Then, thedeletion of the first 52 amino acids was performed using the Q5site-directed mutagenesis kit (New England Biolabs) (G�o–LgB143d). For the �-arrestin 2– based NanoLuc complementa-tion assay, �-arrestin 2 was cloned into pNBe3 vector (PromegaCorp., Madison, WI) with XhoI and EcoRI sites. All constructswere verified by sequencing.

Cell culture and transfection

HEK293 cells were from American Type Culture Collection(Manassas, VA) and grown in Dulbecco’s modified Eagle’smedium (Lonza, Verviers, Belgium) containing 10% fetalbovine serum (International Medical Products, Brussels, Bel-gium), 1% penicillin/streptomycin (Lonza), and 1% L-glutamine(Lonza) at 37 °C with 5% CO2. At 80% confluency, cells weretransfected with XtremeGene 9 (Promega Corp., Madison,WI) in a 3:1 (reagent/DNA) ratio according to the manufactu-rer’s recommendations. A 1:1 ratio (GPCR/G� subunit) wasused with a solution of plasmids diluted 1:10 with emptypcDNA3.1�.

Measurement of GPCR–G protein interaction by NanoLuccomplementation assay

Twenty four hours after transfection of HEK293 cells with a1:1 ratio of GPCR and G� subunit plasmid solutions diluted1:10 with empty pcDNA3.1�, cells were detached from 20- or55-cm2 dishes with trypsin. After one wash with PBS, cells wereresuspended into Hanks’ balanced salt solution (HBSS: 120 mM

NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 10 mM HEPES, pH 7.4)supplemented with the NanoLuc substrate (Promega Corp.,Madison, WI), seeded into a white 96-well plate (50,000 cells/well), and incubated for 45 min at 37 °C. Cells were stimulatedby adding 1 �l of 100 ligand solutions, and luminescence wasrecorded for several minutes depending on each experiment(Centro XS3 LB960; Berthold Technologies). The same proto-col was used for �-arrestin 2 recruitment after a transient trans-fection of receptor and �-arrestin 2– encoding plasmids diluted1:10 with empty pcDNA3.1� into HEK293 cells in a 1:1 ratio.

To determine the Z� factor, cells co-transfected with D2–NPand G�i1–LgBN-term or D2–NP and G�o–LgB143d were resus-pended into HBSS supplemented with NanoLuc substrate andseeded in a white 96-well plate (50,000 cells/well) at 37 °C for 45min. Half of the plate was stimulated with vehicle and the otherhalf with dopamine 100 concentrated, and the luminescencewas recorded for 10 min. The screening of the Prestwick Chem-ical Library� was performed at 5 �M on HEK293 transientlytransfected with the pair of D2–NP and G�i1–LgBN-term orG�o–LgB143d (1:1 ratio with a solution of plasmids diluted 1:10with empty pcDNA3.1�). The cells were detached in parallelfor G�i1 and G�o, resuspended into HBSS supplemented withthe NanoLuc substrate (Promega Corp.), and incubated for 45min at 37 °C. Then, the compounds of the library (dissolved inDMSO) were added into a D2–G�i1 plate, and luminescencewas recorded for 10 min. Next, the compounds of the sameplate of the library were added to the D2–G�o plate, and lumi-nescence was recorded for 10 min.

Data analysis and hit selection criteria

All data show a representative result from three independentexperiments, except data from Fig. 4, which represent themean � S.D. of three independent experiments. Data were col-lected as RLU during 10 min after stimulation, and the areasunder the curve were calculated and then normalized to thevehicle, considered as 0% activity. Statistical significance of theNanoLuc activity in the presence of ligand from basal activitywas assessed using the nonparametric Mann-Whitney test (***,p � 0,001). Concentration-response curves were fitted to thefour-parameter Hill equation using the least-squares method(GraphPad Prism, version 5.0 for Windows; GraphPad, La Jolla,CA). To combine the results obtained for all tested ligands, weconsidered the Emax of dopamine as equal to 100% and repre-sented the activity of each ligand compared with the activity ofdopamine.

The Z� factor was calculated as follows: Z� � 1 �((3�c� � 3�c�)/(��c� � �c��)), where � represents the standard devia-tion; � represents the mean; c�, positive control; and c� neg-ative control. For the screening of the Prestwick ChemicalLibrary�, compounds were considered as hits when the

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ratio � (mean ratio(VEH) � 3 S.D.(VEH)). Ratio representsRLU(compound)/RLU(VEH), and VEH is vehicle. Bias factor wascalculated in two steps. First, we determined the �log(Emax/EC50) for the G�i1 and G�o pathway, which represents the dif-ference between D2 agonist values and dopamine values used asreference ligand obtained with the G�i1- and G�o-based assay.Then, the bias factor corresponding to ��log(Emax/EC50) wascalculated as follows: �log(Emax/EC50)G�i1 pathway � �log(E

max/

EC50)G�o pathway.

Author contributions—C. L., N. D., and J. H. conceptualization; C. L.and J. H. data curation; C. L. and J. H. formal analysis; C. L. and J. H.writing-original draft; C. L., N. D., and J. H. writing-review and edit-ing; N. D. resources; J. H. supervision; J. H. funding acquisition; J. H.investigation; J. H. methodology; J. H. project administration.

Acknowledgments—The Prestwick Chemical Library� used for thescreening was a generous gift from Dr. Jean-Claude Twizere, Labora-tory of Protein Signaling and Interactions, GIGA-Molecular Biologyof Diseases, University of Liège. We acknowledge the technical assis-tance of Céline Piron, and we thank Dr. Andy Chevigné for fruitfuldiscussions and critical reading of the manuscript. We thank theGIGA Imaging and Flow Cytometry Platform.

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Real-time GPCR–G protein interaction assay

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Céline Laschet, Nadine Dupuis and Julien HansonG protein interactions−enables profiling of individual GPCR

A dynamic and screening-compatible nanoluciferase-based complementation assay

doi: 10.1074/jbc.RA118.006231 originally published online December 28, 20182019, 294:4079-4090.J. Biol. Chem. 

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