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.
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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.
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 Figures 1–8, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 1853 kb)
Surface expression and assay validation of Tango constructs (XLSX 20 kb)
Constitutive activity of Tango constructs (XLSX 17 kb)
Screening results of 91 GPCRs at a small compound library (XLSX 689 kb)
Tango assays validated with concentration-response curves (XLSX 20 kb)
Individual concentration-response curves for GPCRs in the Tango assay. (PDF 1459 kb)
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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). https://doi.org/10.1038/nsmb.3014
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