PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome



G protein–coupled receptors (GPCRs) are essential mediators of cellular signaling and are important targets of drug action. Of the approximately 350 nonolfactory human GPCRs, more than 100 are still considered to be 'orphans' because their endogenous ligands remain unknown. Here, we describe a unique open-source resource that allows interrogation of the druggable human GPCRome via a G protein–independent β-arrestin–recruitment assay. We validate this unique platform at more than 120 nonorphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs and describe a method (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation (PRESTO-Tango)) for the simultaneous and parallel interrogation of the entire human nonolfactory GPCRome.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Design, principle and validation of selected Tango assays.
Figure 2: Demonstration of arrestin mobilization with the Tango assay.
Figure 3: Constitutive (or basal) activity for Tango-ized constructs.
Figure 4: Compound profiling with the PRESTO-Tango method at the human GPCRome.
Figure 5: New ligand-target interactions detected by parallel GPCRome screening.
Figure 6: Tango-izing the druggable GPCRome.


  1. 1

    Allen, J.A. & Roth, B.L. Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 51, 117–144 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Bjarnadóttir, T.K. et al. Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse. Genomics 88, 263–273 (2006).

    Article  Google Scholar 

  3. 3

    Fredriksson, R. & Schioth, H.B. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 67, 1414–1425 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Vassilatis, D.K. et al. The G protein-coupled receptor repertoires of human and mouse. Proc. Natl. Acad. Sci. USA 100, 4903–4908 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Hopkins, A.L. & Groom, C.R. The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Rask-Andersen, M., Masuram, S. & Schioth, H.B. The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu. Rev. Pharmacol. Toxicol. 54, 9–26 (2014).

    CAS  Article  Google Scholar 

  7. 7

    Edwards, A.M. et al. Too many roads not taken. Nature 470, 163–165 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Coward, P., Chan, S.D., Wada, H.G., Humphries, G.M. & Conklin, B.R. Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal. Biochem. 270, 242–248 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Eglen, R.M., Bosse, R. & Reisine, T. Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening. Assay Drug Dev. Technol. 5, 425–451 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Emkey, R. & Rankl, N.B. Screening G protein-coupled receptors: measurement of intracellular calcium using the fluorometric imaging plate reader. Methods Mol. Biol. 565, 145–158 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Hill, S.J., Williams, C. & May, L.T. Insights into GPCR pharmacology from the measurement of changes in intracellular cyclic AMP; advantages and pitfalls of differing methodologies. Br. J. Pharmacol. 161, 1266–1275 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Liu, B. & Wu, D. Analysis of the coupling of G12/13 to G protein-coupled receptors using a luciferase reporter assay. Methods Mol. Biol. 237, 145–149 (2004).

    CAS  PubMed  Google Scholar 

  13. 13

    Rodrigues, D.J. & McLoughlin, D. Using reporter gene technologies to detect changes in cAMP as a result of GPCR activation. Methods Mol. Biol. 552, 319–328 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Siehler, S. & Guerini, D. Novel GPCR screening approach: indirect identification of S1P receptor agonists in antagonist screening using a calcium assay. J. Recept. Signal Transduct. Res. 26, 549–575 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Lefkowitz, R.J. & Shenoy, S.K. Transduction of receptor signals by beta-arrestins. Science 308, 512–517 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Roth, B.L. & Marshall, F.H. NOBEL 2012 Chemistry: studies of a ubiquitous receptor family. Nature 492, 57 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Barak, L.S., Ferguson, S.S., Zhang, J. & Caron, M.G. A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J. Biol. Chem. 272, 27497–27500 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  Google Scholar 

  19. 19

    Yan, Y.X. et al. Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology. J. Biomol. Screen. 7, 451–459 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl. Acad. Sci. USA 105, 64–69 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Fang, Y., Li, G. & Ferrie, A.M. Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells. J. Pharmacol. Toxicol. Methods 55, 314–322 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Hennen, S. et al. Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist. Sci. Signal. 6, ra93 (2013).

    Article  Google Scholar 

  23. 23

    Guan, X.M., Kobilka, T.S. & Kobilka, B.K. Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. J. Biol. Chem. 267, 21995–21998 (1992).

    CAS  PubMed  Google Scholar 

  24. 24

    Hanson, B.J. et al. A homogeneous fluorescent live-cell assay for measuring 7-transmembrane receptor activity and agonist functional selectivity through beta-arrestin recruitment. J. Biomol. Screen. 14, 798–810 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Kim, K.M. & Caron, M.G. Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and beta-arrestin interaction. Biochem. Biophys. Res. Commun. 366, 42–47 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Vrecl, M. et al. Beta-arrestin-based Bret2 screening assay for the “non”-beta-arrestin binding CB1 receptor. J. Biomol. Screen. 14, 371–380 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Cho, D.I., Beom, S., Van Tol, H.H., Caron, M.G. & Kim, K.M. Characterization of the desensitization properties of five dopamine receptor subtypes and alternatively spliced variants of dopamine D2 and D4 receptors. Biochem. Biophys. Res. Commun. 350, 634–640 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Jensen, D.D. et al. The bile acid receptor TGR5 does not interact with beta-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts. J. Biol. Chem. 288, 22942–22960 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Goupil, E. et al. Biasing the prostaglandin F2α receptor responses toward EGFR-dependent transactivation of MAPK. Mol. Endocrinol. 26, 1189–1202 (2012).

    CAS  Article  Google Scholar 

  30. 30

    Tulipano, G. et al. Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes. J. Biol. Chem. 279, 21374–21382 (2004).

    CAS  Article  Google Scholar 

  31. 31

    Richard, F., Barroso, S., Martinez, J., Labbe-Jullie, C. & Kitabgi, P. Agonism, inverse agonism, and neutral antagonism at the constitutively active human neurotensin receptor 2. Mol. Pharmacol. 60, 1392–1398 (2001).

    CAS  Article  Google Scholar 

  32. 32

    Vita, N. et al. Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells. Eur. J. Pharmacol. 360, 265–272 (1998).

    CAS  Article  Google Scholar 

  33. 33

    Keiser, M.J. et al. Predicting new molecular targets for known drugs. Nature 462, 175–181 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Auld, D.S. et al. Characterization of chemical libraries for luciferase inhibitory activity. J. Med. Chem. 51, 2372–2386 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Auld, D.S., Thorne, N., Maguire, W.F. & Inglese, J. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc. Natl. Acad. Sci. USA 106, 3585–3590 (2009).

    CAS  Article  Google Scholar 

  36. 36

    Auld, D.S., Thorne, N., Nguyen, D.T. & Inglese, J. A specific mechanism for nonspecific activation in reporter-gene assays. ACS Chem. Biol. 3, 463–470 (2008).

    CAS  Article  Google Scholar 

  37. 37

    Southern, C. et al. Screening beta-arrestin recruitment for the identification of natural ligands for orphan G-protein-coupled receptors. J. Biomol. Screen. 18, 599–609 (2013).

    Article  Google Scholar 

  38. 38

    Allen, J.A. et al. Discovery of beta-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc. Natl. Acad. Sci. USA 108, 18488–18493 (2011).

    CAS  Article  Google Scholar 

  39. 39

    Fenalti, G. et al. Molecular control of δ-opioid receptor signalling. Nature 506, 191–196 (2014).

    CAS  Article  Google Scholar 

  40. 40

    White, K.L. et al. Identification of novel functionally selective kappa-opioid receptor scaffolds. Mol. Pharmacol. 85, 83–90 (2014).

    Article  Google Scholar 

  41. 41

    Wu, H. et al. Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012).

    CAS  Article  Google Scholar 

  42. 42

    Wacker, D. et al. Structural features for functional selectivity at serotonin receptors. Science 340, 615–619 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Stanasila, L., Abuin, L., Dey, J. & Cotecchia, S. Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50. Mol. Pharmacol. 74, 562–573 (2008).

    CAS  Article  Google Scholar 

  44. 44

    Turu, G. et al. Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors. FEBS Lett. 580, 41–45 (2006).

    CAS  Article  Google Scholar 

  45. 45

    Cao, W. et al. Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation. J. Biol. Chem. 275, 38131–38134 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Solinski, H.J., Gudermann, T. & Breit, A. Pharmacology and signaling of MAS-related G protein-coupled receptors. Pharmacol. Rev. 66, 570–597 (2014).

    Article  Google Scholar 

  47. 47

    Liu, Q. et al. Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 139, 1353–1365 (2009).

    Article  Google Scholar 

  48. 48

    Twaites, B., Wilton, L.V., Layton, D. & Shakir, S.A. Safety of nateglinide as used in general practice in England: results of a prescription-event monitoring study. Acta Diabetol. 44, 233–239 (2007).

    CAS  Article  Google Scholar 

  49. 49

    Katritch, V., Cherezov, V. & Stevens, R.C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012).

    CAS  Article  Google Scholar 

  50. 50

    Jordan, M., Schallhorn, A. & Wurm, F.M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996).

    CAS  Article  Google Scholar 

Download references


This work was supported by US National Institutes of Health grant R01DA27170 (B.L.R., M.F.S. and W.K.K.) and UO1MH104974 (B.L.R., W.K.K., M.F.S. and K.L.), the US National Institute of Mental Health Psychoactive Drug Screening Program (B.L.R., M.F.S., W.K.K. and X.-P.H.) and the Michael Hooker Distinguished Professorship (B.L.R.). K.L. was supported by the University of North Carolina Department of Pharmacology Training Program (NIH 5-T32-GM007040). The authors thank R. Stevens and S. Katrich for allowing us to use and modify their GPCRome tree (Fig. 6) from ref. 49. We thank R. Axel (Columbia University) for providing HTLA cells.

Author information




B.L.R. and W.K.K. conceived the general approach; W.K.K. designed the constructs; W.K.K., M.F.S., K.L. and X.-P.H. executed and analyzed validation, profiling and confirmatory assays; J.D.M. and P.M.G. validated assays; N.S. assisted with high-content microscopy; M.F.S. designed, executed and analyzed the simultaneous profiling strategy; B.L.R., W.K.K., M.F.S., K.L. and X.-P.H. wrote the paper; B.L.R. was responsible for the overall strategy.

Corresponding author

Correspondence to Bryan L Roth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Demonstration of the feasibility of testing for antagonist activity with the Tango β-arrestin recruitment assay and demonstration of the variability of the effects of removal of the V2 tail on activity in the Tango β-arrestin recruitment assay.

(a-c) Demonstration of the feasibility of testing for antagonist activity with the Tango β-arrestin–recruitment assay (a) Stimulation of neurotensin receptor NTSR2 activity by SR48692 or SR142948, but not neurotensin. (b) Inhibition of activity of SR48692 at the NTSR2 receptor by levocabastine or neurotensin. (c) Inhibition of activity of SR142948 at the NTSR2 receptor by levocabastine or neurotensin. (d-f) Demonstration of the variability of the effects of removal of the V2 tail on activity in the Tango β-arrestin recruitment assay. (d) Lack of effect of V2 tail removal using the LTB4R receptor. (e) Increased activity after V2 tail removal using the CMKLR1 receptor. (f) Decreased activity after V2 tail removal using the FFAR2 (GPR43) receptor.

Supplementary Figure 2 The effect of clozapine on concentration-response curves to LSD at various GPCR targets in the Tango β-arrestin recruitment assay.

Data are shown as mean ± SEM of quadruplicate values, and curves were fitted using GraphPad Prism. (a) HTR1A, (b) HTR1D, (c) HTR1B, (d) ADRA2B, (e) HTR1E, (f) HTR1F, (g) HTR2A, (h) HTR5, (i) DRD2.

Supplementary Figure 3 Summary of constitutive activity of GPCR-Tango constructs used in this study.

HTLA cells were transfected with various Tango constructs, plated into 384-well assay plates, and luminescence in relative luminescence units (RLU) was measured after overnight incubation in the absence of ligand. The constitutive activity of these constructs, i.e., the ratio of the maximum to the minimum luminescence of the constructs, varied over a range of up to 551-fold in individual experiments.

Supplementary Figure 4 The effect of various lengths of exposure to agonist on the luminescence response in the Tango β-arrestin recruitment assay.

Cells transfected with the DRD2-Tango construct were plated into 384-well plates and incubated in medium containing 1% dialysed fetal bovine serum (dFBS) overnight. Then, medium was switched to serum-free, and cells were incubated for a further 4 hours. Various concentrations of the agonist quinpirole were added, and at different times were washed out and replaced with serum-free medium, with further incubation overnight. Plates were read the following day. Data are expressed as mean ± SEM of quadruplicate determinations, and curves were fitted using GraphPad Prism.

Supplementary Figure 5 Concentration-response curves of nateglinide in Gs and Gi assays in MRGPRX receptor–expressing cells.

a) Gs response to nateglinide in HEK293T cells expressing MRGPRX receptors, showing a Gs response only in cells expressing MRGPRX4 receptors at high concentrations of nateglinide. (b) Comparison of the concentration-response curves in Gs assays of nateglinide and isoproterenol in MRGPRX4-expressing HEK293T cells. (c) Concentration-response curve in Gi assay of nateglinide in MRGPRX4-expressing HEK293T cells. Data are expressed as mean ± SEM of triplicate or quadruplicate determinations, and curves were fitted using Graphpad Prism.

Supplementary Figure 6 Calcium-mobilization responses in stable cell lines expressing MRGPRX receptors.

(a,c,e) Concentration-response curves; data are expressed as mean ± SEM, and curves were fitted using Graphpad Prism. (b,d,f) Time course of responses, showing representative curves of experiments done in triplicate. (a, b) Responses of MRGPRX1-expressing cells to BAM8-22. (c,d) Responses of MRGPRX2-expressing cells to SB 205,607. (e,f) Responses of MRGPRX4-expressing cells to nateglinide. TRAP is an agonist for endogenous PAR1 and serves as an internal control for the calcium mobilization assay.

Supplementary Figure 7 Concentration-response curves showing responses of bombesin receptors to the cognate ligand bombesin and saquinavir.

(a) BB1 receptor, Tango assay. (b) BB2 receptor, Tango assay. (c) BB1 receptor, calcium mobilization assay. (d) BB2 receptor, calcium mobilization assay. (e) BB3 receptor, PI hydrolysis assay. Data are expressed as mean ± SEM of triplicate determinations, and curves were fitted using Graphpad Prism.

Supplementary Figure 8 Concentration-dependent agonist activity of various compounds at MRGPRX2 receptors in the Tango assay compared with the activity of the known ligand SB 205607.

(a) and (c) Tango arrestin recruitment assays, (b) PI hydrolysis. Data are expressed as mean ± SEM of triplicate determinations, and curves were fitted using Graphpad Prism.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 1853 kb)

Supplementary Table 2

Surface expression and assay validation of Tango constructs (XLSX 20 kb)

Supplementary Table 3

Constitutive activity of Tango constructs (XLSX 17 kb)

Supplementary Table 4

Screening results of 91 GPCRs at a small compound library (XLSX 689 kb)

Supplementary Table 5

Tango assays validated with concentration-response curves (XLSX 20 kb)

Supplementary Data Set 1

Individual concentration-response curves for GPCRs in the Tango assay. (PDF 1459 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kroeze, W., Sassano, M., Huang, XP. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol 22, 362–369 (2015).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing