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.

  • Protocol
  • Published:

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

Nature Protocols volume 3, pages 1402–1413 (2008)Cite this article

Abstract

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant–OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.

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: Overview of the functional activation of ORs in a heterologous expression system.
Figure 2: The endogenous and heterologous OR signal transduction pathways.
Figure 3
Figure 4: An example of cell-surface OR expression.
Figure 5: An example of cell-surface flow cytometry.
Figure 6: A hypothetical case of luciferase assay data interpretation.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Barnea, G. et al. Odorant receptors on axon termini in the brain. Science 304, 1468 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Strotmann, J., Levai, O., Fleischer, J., Schwarzenbacher, K. & Breer, H. Olfactory receptor proteins in axonal processes of chemosensory neurons. J. Neurosci. 24, 7754–7761 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Niimura, Y. & Nei, M. Extensive gains and losses of olfactory receptor genes in mammalian evolution. PLoS ONE 2, e708 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Niimura, Y. & Nei, M. Evolutionary changes of the number of olfactory receptor genes in the human and mouse lineages. Gene 346, 23–28 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Godfrey, P.A., Malnic, B. & Buck, L.B. The mouse olfactory receptor gene family. Proc. Natl. Acad. Sci. USA 101, 2156–2161 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Malnic, B., Godfrey, P.A. & Buck, L.B. The human olfactory receptor gene family. Proc. Natl. Acad. Sci. USA 101, 2584–2589 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Zhang, X. & Firestein, S. The olfactory receptor gene superfamily of the mouse. Nat. Neurosci. 5, 124–133 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Glusman, G., Yanai, I., Rubin, I. & Lancet, D. The complete human olfactory subgenome. Genome Res. 11, 685–702 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Young, J.M. et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families. Hum. Mol. Genet. 11, 535–546 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Amoore, J.E., Johnston, J.W. Jr. & Rubin, M. The Sterochemical theory of odor. Sci. Am. 210, 42–49 (1964).

    Article  CAS  PubMed  Google Scholar 

  11. Buck, L. & Axel, R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175–187 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Tate, C.G. & Grisshammer, R. Heterologous expression of G-protein-coupled receptors. Trends Biotechnol. 14, 426–430 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Lu, M., Echeverri, F. & Moyer, B.D. Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors. Traffic 4, 416–433 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. McClintock, T.S. et al. Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors. Brain Res. Mol. Brain Res. 48, 270–278 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Gimelbrant, A.A., Haley, S.L. & McClintock, T.S. Olfactory receptor trafficking involves conserved regulatory steps. J. Biol. Chem. 276, 7285–7290 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Dwyer, N.D., Troemel, E.R., Sengupta, P. & Bargmann, C.I. Odorant receptor localization to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein. Cell 93, 455–466 (1998).

    Article  CAS  Google Scholar 

  17. Saito, H., Kubota, M., Roberts, R.W., Chi, Q. & Matsunami, H. RTP family members induce functional expression of mammalian odorant receptors. Cell 119, 679–691 (2004).

    Article  CAS  Google Scholar 

  18. Zhuang, H. & Matsunami, H. Synergism of accessory factors in functional expression of mammalian odorant receptors. J. Biol. Chem. 282, 15284–15293 (2007).

    Article  CAS  Google Scholar 

  19. Von Dannecker, L.E., Mercadante, A.F. & Malnic, B. Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the olfactory-specific G-protein Galphaolf. J. Neurosci. 25, 3793–3800 (2005).

    Article  PubMed  Google Scholar 

  20. Von Dannecker, L.E., Mercadante, A.F. & Malnic, B. Ric-8B promotes functional expression of odorant receptors. Proc. Natl. Acad. Sci. USA 103, 9310–9314 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Keller, A., Zhuang, H., Chi, Q., Vosshall, L.B. & Matsunami, H. Genetic variation in a human odorant receptor alters odour perception. Nature 449, 468–472 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Katada, S., Nakagawa, T., Kataoka, H. & Touhara, K. Odorant response assays for a heterologously expressed olfactory receptor. Biochem. Biophys. Res. Commun. 305, 964–969 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Sands, W.A. & Palmer, T.M. Regulating gene transcription in response to cyclic AMP elevation. Cell Signal 20, 460–466 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Zhao, H. et al. Functional expression of a mammalian odorant receptor. Science 279, 237–242 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Touhara, K. et al. Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proc. Natl. Acad. Sci. USA 96, 4040–4045 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Krautwurst, D., Yau, K.W. & Reed, R.R. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95, 917–926 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Kajiya, K. et al. Molecular bases of odor discrimination: reconstitution of olfactory receptors that recognize overlapping sets of odorants. J. Neurosci. 21, 6018–6025 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Offermanns, S. & Simon, M.I. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175–15180 (1995).

    Article  CAS  PubMed  Google Scholar 

  29. Shirokova, E. et al. Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J. Biol. Chem. 280, 11807–11815 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Chandrashekar, J. et al. T2Rs function as bitter taste receptors. Cell 100, 703–711 (2000).

    Article  CAS  Google Scholar 

  31. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

    Google Scholar 

  32. Harlow, E., Lane, D. & Cold Spring Harbor Laboratory Antibodies: a Laboratory Manual xiii, 726 p. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

    Google Scholar 

  33. Araki, I. & Nakamura, H. Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate. Development 126, 5127–5135 (1999).

    CAS  PubMed  Google Scholar 

  34. Laird, D.W. & Molday, R.S. Evidence against the role of rhodopsin in rod outer segment binding to RPE cells. Invest. Ophthalmol. Vis. Sci. 29, 419–428 (1988).

    CAS  PubMed  Google Scholar 

  35. Osborn, M. & Weber, K. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 24, 97–132 (1982).

    Article  CAS  PubMed  Google Scholar 

  36. Philpott, N.J. et al. The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques. Blood 87, 2244–2251 (1996).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Toyama for FACS data. This research was supported by grants to H.M. from the NIH and Human Frontier Science Program and to H.Z. from an NIH NRSA Predoctoral fellowship.

Author information

Author notes

  1. Hiroaki Matsunami

    Present address: Department of Neurobiology, Duke University Medical Center, Durham, 27710, North Carolina, USA

  2. Hanyi Zhuang

    Present address: Current address: Department of Pathophysiology, Shanghai Jiatong University School of Medicine, Shanghai 200025, PRC.,

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Hanyi Zhuang & Hiroaki Matsunami

Authors
  1. Hanyi Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroaki Matsunami
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hanyi Zhuang or Hiroaki Matsunami.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhuang, H., Matsunami, H. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc 3, 1402–1413 (2008). https://doi.org/10.1038/nprot.2008.120

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.120

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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