Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

The use of constitutively active GPCRs in drug discovery and functional genomics

Nature Reviews Drug Discovery volume 1, pages 599–608 (2002)Cite this article

Key Points

  • 'Orphan' G-protein-coupled receptors (GPCRs) — those for which endogenous ligands remain to be identified — offer great promise for the detection of novel GPCR-based therapeutics and have been an increasing focus for the pharmaceutical industry.

  • However, traditional drug screening at GPCRs has relied on the identification of small molecules that interfere with ligand binding to GPCRs. So, the large number of orphan GPCRs identified from genomic studies for which no ligand is available has precipitated several approaches to ligand discovery as a considered prerequisite to drug screening.

  • One such approach involves the use of constitutively active GPCRs, which stimulate cellular signalling pathways in the absence of ligand attachment.

  • The precise mechanism for constitutive activity remains to be established, but it is probable that changes in the core seven-transmembrane-domain (7TM) region of GPCRs underlies constitutive activation.

  • Changes in the amino-acid composition of a 'latch' region in GPCRs can cause alterations in receptor conformation that result in constitutive activity. This forms the basis of constitutively activating receptor technology (CART), which opens up orphan, as well as known, GPCR families to ligand-independent small-molecule screening approaches.

  • Constitutively active GPCRs can be used in various detection platforms for high-throughput screening; for example, using methods that detect cAMP production, including alpha screen, flash plate or enzyme-linked immunosorbent assay (for receptors that couple to Gi or Gs), or that detect calcium flux or inositol phosphate production (for receptors that couple to Gq).

  • Furthermore, constitutively active receptors can also be used in small-molecule screens of transfected melanophore cells, which contain pigmented organelles that disperse or aggregate in response to selective GPCR activity. This visually based bioassay is applicable across the GPCR family.

Abstract

The complete sequencing of the human genome has afforded researchers the opportunity to identify novel G-protein-coupled receptors (GPCRs) that are expressed in human tissues. The successful identification of hundreds of GPCRs represents the single greatest opportunity for novel drug development today. However, the lack of identified ligands for these GPCRs has limited their utility for traditional drug discovery approaches that focus on ligand-based assay methods to discover and pharmacologically characterize drug candidates. Here, we review the use of constitutively activated GPCRs in the discovery pathway, both as a means to overcome the limitations of traditional drug discovery at novel GPCRs and as a tool to investigate the functionality of these receptors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Family classification of known G-protein-coupled receptors.
Figure 2: G-protein-coupled receptor (GPCR) family classification of known and orphan (non-'olfactory') GPCRs.
Figure 3: Representation of 'inactive' and 'active' GPCR conformations.
Figure 4: The CART α2-adrenoceptor responds better to an agonist and to inverse agonists than the wild-type receptor.
Figure 5: Cluster analysis of GPCR gene expression levels over 80 human peripheral and neuronal tissues.
Figure 6: The use of a constitutively active orphan GPCR to delineate in vitro functionality.

Similar content being viewed by others

References

  1. Marinissen, M. J. & Gutkind, J. G. G protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol. Sci. 22, 368–376 (2001).

    Article  CAS  Google Scholar 

  2. Scussa, F. World's best-selling drugs. Med Ad News 21 No. 5, 1–46 (2002).

    Google Scholar 

  3. Marchese, A., George, S. R., Kolakowski, L. F., Lynch, K. R. & O'Dowd, B. F. Novel GPCRs and their endogenous ligands: expanding the boundaries of physiology and pharmacology. Trends Pharmacol. Sci. 20, 370–375 (1999).

    Article  CAS  Google Scholar 

  4. Venter, J. C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    Article  CAS  Google Scholar 

  5. Menzaghi, F., Behan, D. P. & Chalmers, D. T. Constitutively activated G protein-coupled receptors: a novel approach to CNS drug discovery. Curr. Drug Targets 1, 105–121 (2002).

    CAS  Google Scholar 

  6. Civelli, O. et al. Novel neurotransmitters as natural ligands of orphan G protein-coupled receptors. Trends Neurosci. 24, 230–237 (2001).This review summarizes ligand identification at orphan GPCRs.

    Article  CAS  Google Scholar 

  7. Bunzow, JR. et al. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783–787 (1988).

    Article  CAS  Google Scholar 

  8. Meng, F. et al. Cloning and expression of the A2a adenosine receptor from guinea pig brain. Neurochem. Res. 19, 615–623 (1994).

    Article  Google Scholar 

  9. Lovenberg, T. W. et al. Cloning and characterization of a functionally distinct corticotrophin-releasing factor receptor subtype from rat brain. Proc. Natl Acad. Sci. USA 92, 836–840 (1995).

    Article  CAS  Google Scholar 

  10. Takeda, S., Kadowaki, S., Haga, T., Takaesu, H. & Mitaku, S. Identification of G protein-coupled receptor genes from the human genome sequence. FEBS Lett. 520, 97–101 (2002).

    Article  CAS  Google Scholar 

  11. Karchin, R., Karplus, K. & Haussler, D. Classifying G-protein coupled receptors with support vector machines. Bioinformatics 18, 147–159 (2002).

    Article  CAS  Google Scholar 

  12. Durant, G. J., Parsons, M. E. & Black, J. W. Potential histamine H2-receptor antagonists. 2. N-α-guanylhistamine. J. Med. Chem. 18, 830–833 (1975).

    Article  CAS  Google Scholar 

  13. Wilson, S. et al. Orphan G-protein-coupled receptors: the next generation of drug targets? Br. J. Pharmacol. 125, 1387–1392 (1998).

    Article  CAS  Google Scholar 

  14. Meunier, J. C. et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377, 532–535 (1995).

    Article  CAS  Google Scholar 

  15. Sakurai, T. et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573–585 (1998).

    Article  CAS  Google Scholar 

  16. Hinuma, S. et al. A prolactin-releasing peptide in the brain. Nature 393, 272–276 (1998).

    Article  CAS  Google Scholar 

  17. Tatemoto, K. et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun. 251, 471–476 (1998).

    Article  CAS  Google Scholar 

  18. Kojima, M. et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660 (1999).

    Article  CAS  Google Scholar 

  19. Erickson, J. R. et al. Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid. J. Biol. Chem. 273, 1506–1510 (1998).

    Article  CAS  Google Scholar 

  20. Heise, C. E. et al. Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem. 275, 30531–30536 (2000).

    Article  CAS  Google Scholar 

  21. Chambers, J. et al. Melanin-concentrating hormone is the cognate ligand for the orphan G protein-coupled receptor SLC-1. Nature 400, 261–265 (1999).

    Article  CAS  Google Scholar 

  22. Mori, M. et al. Urotensin II is the endogenous ligand of a G protein-coupled orphan receptor, SENR (GPR14). Biochem Biophys. Res. Commun. 265, 123–129 (1999).

    Article  CAS  Google Scholar 

  23. Feighner, S. D. et al. Receptor for Motilin identified in the human gastrointestinal system. Science 284, 2184–2188 (1999).

    Article  CAS  Google Scholar 

  24. Kojima, M. et al. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem. Biophys. Res. Commun. 276, 435–438 (2000).

    Article  CAS  Google Scholar 

  25. Elshourbagy, N. A. et al. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J. Biol. Chem. 275, 25965–25971 (2000).

    Article  CAS  Google Scholar 

  26. Lembo, P. M. C. et al. Proenkephalin A gene products activate a new family of sensory neuron-specific GPCRs. Nature Neurosci. 5, 201–209 (2002).

    Article  CAS  Google Scholar 

  27. Masuda, Y. et al. Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors. Biochem. Biophys. Res. Commun. 293, 396–402 (2002).

    Article  CAS  Google Scholar 

  28. Costa, T. & Hertz, A. Antagonists with negative intrinsic activity at α-opioid receptors coupled to GTP-binding proteins. Proc. Natl Acad. Sci. USA 86, 7321–7325 (1989).

    Article  CAS  Google Scholar 

  29. Samama, P., Cotecchia, S., Costa, T. & Lefkowitz, R. J. A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 268, 4625–4636 (1993).This article describes the importance of the allosteric ternary complex model for GPCR action.

    CAS  PubMed  Google Scholar 

  30. Lefkowitz, R. J., Cotecchia, S., Samama, P. & Costa, T. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol. Sci. 14, 303–307 (1993).

    Article  CAS  Google Scholar 

  31. Parma, J. et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365, 649–651 (1993).

    Article  CAS  Google Scholar 

  32. Parfitt, A. M. et al. Hypercalcemia due to constitutive activity of the parathyroid hormone (PTH/PTH-related) receptor: comparison with primary hyperparathyroidism. J. Clin. Endocrinol. Metab. 81, 3584–3588 (1996).

    CAS  PubMed  Google Scholar 

  33. Balmforth, A. J., Lee, A. J., Warburton, P., Donnelly, D. & Ball, S. G. The conformational change responsible for the AT1 receptor activation is dependent upon two juxtaposed asparagine residues on transmembrane helices III and VII. J. Biol. Chem. 272, 4245–4251 (1997).

    Article  CAS  Google Scholar 

  34. Alewijnse, A. E. et al. The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H (2) receptor. Mol. Pharmacol. 57, 890–898 (2000).

    CAS  PubMed  Google Scholar 

  35. Huang, P. et al. Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp. Biochemistry 40, 13501–13509 (2001).

    Article  CAS  Google Scholar 

  36. Chen, S., Lin, F. & Graham, R. M. Phe(303) in TMVI of the α1B-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation. Biochemistry 41, 588–596 (2002).

    Article  CAS  Google Scholar 

  37. Behan, D. P. & Chalmers, D. T. The use of constitutively active receptors for drug discovery at the G protein-coupled receptor gene pool. Curr. Opin. Drug Discov. Dev. 4, 548–560 (2001).

    CAS  Google Scholar 

  38. Parnot, C. et al. Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay. Proc. Natl Acad. Sci. USA 97, 7615–7620 (2000).This paper illustrates the potential for mutation-induced alterations in GPCR constitutive activity.

    Article  CAS  Google Scholar 

  39. Nakabayashi, K., Kudo, M., Kobilka, B. & Hsueh, A. J. W. Activation of lutenizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region. J. Biol. Chem. 275, 30264–30271 (2000).

    Article  CAS  Google Scholar 

  40. Rasmussen, S. G. F. et al. Mutation of a highly conserved aspartic acid in the β2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6. Mol. Pharmacol. 56, 175–184 (1999).

    Article  CAS  Google Scholar 

  41. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    Article  CAS  Google Scholar 

  42. Lu, Z. L., Saldanha, J. W. & Hulme, E. C. Seven-transmembrane receptors: crystals clarify. Trends Pharmacol. Sci. 23, 140–146 (2002).This review summarizes crystallization data related to conformational changes of GPCRs.

    Article  CAS  Google Scholar 

  43. Behan, D. P. & Chalmers, D. T. A method of identifying modulators of cell surface membrane receptors useful in the treatment of disease. WO Patent 9,846,995 (1997).

  44. Lerner, M. R. Tools for investigating functional interactions between ligands and G protein-coupled receptors. Trends Neurosci. 17, 142–146 (1994).This article reviews the application of melanophore technology for GPCR screening.

    Article  CAS  Google Scholar 

  45. Chen, G. et al. Use of constitutive G protein-coupled receptor activity for drug discovery. Mol. Pharmacol. 57, 125–134 (2000).

    Article  CAS  Google Scholar 

  46. Strange, P. G. Mechanisms of inverse agonism at G protein-coupled receptors. Trends Pharmacol. Sci. 23, 89–95 (2002).This article discusses mechanisms that could explain inverse-agonist action.

    Article  CAS  Google Scholar 

  47. Kenakin, T. Efficacy at G-protein-coupled receptors. Nature Rev. Drug Discov. 1, 103–110 (2002).

    Article  CAS  Google Scholar 

  48. Kenakin, T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J. 15, 598–611 (2001).

    Article  CAS  Google Scholar 

  49. Milligan, G., Bond, R. A. & Lee, M. Inverse agonism: pharmacological curiosity or potential therapeutic strategy. Trends Pharmacol. Sci. 16, 10–13 (1995).

    Article  CAS  Google Scholar 

  50. Smit, M. J. et al. Inverse agonism of histamine H2 antagonists accounts for upregulation of spontaneously active histamine H2 receptors. Proc. Natl Acad. Sci. USA 93, 6802–6807 (1996).

    Article  CAS  Google Scholar 

  51. Morisset, S. et al. High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature 408, 860–864 (2000).

    Article  CAS  Google Scholar 

  52. Carroll, F. Y. et al. BAY-36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity. Mol. Pharmacol. 59, 965–973 (2001).

    Article  CAS  Google Scholar 

  53. Fawzi, A. B. et al. SCH-202676: an allosteric modulator of both agonist and antagonist binding to G protein-coupled receptors. Mol. Pharmacol. 59, 30–37 (2001).

    Article  CAS  Google Scholar 

  54. Wada, M., Nagano, N. & Nemeth, E. F. The calcium receptor and calcimimetics. Curr. Opin. Nephrol. Hypertens. 8, 429–433 (1999).

    Article  CAS  Google Scholar 

  55. Soudijn, W., van Wijngaarden, I. & Izerman, A. P. Allosteric modulation of G-protein-coupled receptors. Exp. Opin. Therap. Patents 11, 1889–1904 (2001).This article is a review of small-molecule allosteric modulators that regulate the effects of agonists and antagonists at GPCRs.

    Article  CAS  Google Scholar 

  56. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

  57. Krogh, A., Brown, M., Mian, I. S., Sjolander, K. & Haussler, D. Hidden Markov models in computational biology: application to protein modeling. J. Mol. Biol. 235, 1501–1531 (1994).

    Article  CAS  Google Scholar 

  58. Karplus, K., Barrett, C. & Hughey, R. Hidden Markov models for detecting remote protein homologies. Bioinformatics 14, 846–856 (1998).This article discusses the use of HMMs for identifying members of protein families.

    Article  CAS  Google Scholar 

  59. Babinet, C. & Cohen-Tannoudji, M. Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology. An. Acad. Bras. Cienc. 73, 365–383 (2001).

    Article  CAS  Google Scholar 

  60. Harris, S. & Foord, S. M. Transgenic gene knock-outs: functional genomics and therapeutic target selection. Pharmacogenomics 1, 433–443 (2000).

    Article  CAS  Google Scholar 

  61. Sautel, M. & Milligan, G. Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery. Curr. Med. Chem. 7, 889–896 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge the outstanding technical and intellectual contribution of Arena scientists to this article.

Author information

Authors and Affiliations

  1. Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, 92121, California, USA

    Derek T. Chalmers & Dominic P. Behan

Authors
  1. Derek T. Chalmers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dominic P. Behan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Derek T. Chalmers.

Related links

Related links

DATABASES

LocusLink

α2-adrenoceptors

apelin

glucagon

5-HT2A receptor

insulin

motilin

neuromedin U

neuropeptide FF

rhodopsin

urotensin II

OMIM

hyperthyroidism

Glossary

BIOGENIC AMINES

Prominent among biogenic amines are neurotransmitters, such as serotonin, histamine, dopamine and noradrenaline.

HYPERMORPHIC MUTATION

A mutation that confers an increase in gene function.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chalmers, D., Behan, D. The use of constitutively active GPCRs in drug discovery and functional genomics. Nat Rev Drug Discov 1, 599–608 (2002). https://doi.org/10.1038/nrd872

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd872

This article is cited by

Search

Advanced search

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