Movatterモバイル変換

[0]ホーム
URL:

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.

Advertisement

Nature Methods
  • Article
  • Published:

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Nature Methodsvolume 3, pages1001–1006 (2006)Cite this article

Abstract

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein–coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.Please visit methagora to view and post comments on this article

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

Access options

Access through your institution

Subscription info for Japanese customers

We have a dedicated website for our Japanese customers. Please go tonatureasia.com to subscribe to this journal.

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF

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

Figure 1: Effects of acceptor/donor ratio and surface density on BRETeff.
Figure 2: Monomeric proteins expressed at the cell surface give substantial resonance energy transfer in conventional BRET experiments.
Figure 3: Type-1 experiments: varying the acceptor/donor ratio distinguishes between BRET arising from random versus oligomeric interactions.
Figure 4: Type-2 experiments: BRET signals arising from random interactions of molecules are linearly related to surface density at a given acceptor/donor ratio.
Figure 5: Two native class-A GPCRs are monomeric at the cell surface.

Similar content being viewed by others

References

  1. Xu, Y., Piston, D.W. & Johnson, C.H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.Proc. Natl. Acad. Sci. USA96, 151–156 (1999).

    Article CAS  Google Scholar 

  2. Wolber, P.K. & Hudson, B.S. An analytic solution to the Forster energy transfer problem in two dimensions.Biophys. J.28, 197–210 (1979).

    Article CAS  Google Scholar 

  3. Fung, B.K. & Stryer, L. Surface density determination in membranes by fluorescence energy transfer.Biochemistry17, 5241–5248 (1978).

    Article CAS  Google Scholar 

  4. Veatch, W. & Stryer, L. The dimeric nature of the gramicidin A transmembrane channel: conductance and fluorescence energy transfer studies of hybrid channels.J. Mol. Biol.113, 89–102 (1977).

    Article CAS  Google Scholar 

  5. Kenworthy, A.K. & Edidin, M. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.J. Cell Biol.142, 69–84 (1998).

    Article CAS  Google Scholar 

  6. Pfleger, K.D. & Eidne, K.A. Monitoring the formation of dynamic G-protein-coupled receptor-protein complexes in living cells.Biochem. J.385, 625–637 (2005).

    Article CAS  Google Scholar 

  7. Jones, E.Y., Davis, S.J., Williams, A.F., Harlos, K. & Stuart, D.I. Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2.Nature360, 232–239 (1992).

    Article CAS  Google Scholar 

  8. Bodian, D.L., Jones, E.Y., Harlos, K., Stuart, D.I. & Davis, S.J. Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.Structure2, 755–766 (1994).

    Article CAS  Google Scholar 

  9. Collins, A.V. et al. The interaction properties of costimulatory molecules revisited.Immunity17, 201–210 (2002).

    Article CAS  Google Scholar 

  10. Ikemizu, S. et al. Structure and dimerization of a soluble form of B7–1.Immunity12, 51–60 (2000).

    Article CAS  Google Scholar 

  11. Stamper, C.C. et al. Crystal structure of the B7–1/CTLA-4 complex that inhibits human immune responses.Nature410, 608–611 (2001).

    Article CAS  Google Scholar 

  12. Barclay, A.N. et al.The Leucocyte Antigen Factsbook (Academic Press, San Diego, 1997).

  13. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived fromDiscosoma sp. red fluorescent protein.Nat. Biotechnol.22, 1567–1572 (2004).

    Article CAS  Google Scholar 

  14. Mercier, J.F., Salahpour, A., Angers, S., Breit, A. & Bouvier, M. Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer.J. Biol. Chem.277, 44925–44931 (2002).

    Article CAS  Google Scholar 

  15. Bhatia, S., Edidin, M., Almo, S.C. & Nathenson, S.G. Different cell surface oligomeric states of B7–1 and B7–2: implications for signaling.Proc. Natl. Acad. Sci. USA102, 15569–15574 (2005).

    Article CAS  Google Scholar 

  16. Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).Proc. Natl. Acad. Sci. USA97, 3684–3689 (2000).

    CAS  Google Scholar 

  17. Ramsay, D., Kellett, E., McVey, M., Rees, S. & Milligan, G. Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences.Biochem. J.365, 429–440 (2002).

    Article CAS  Google Scholar 

  18. Chen, Y., Couvineau, A., Laburthe, M. & Amiranoff, B. Solubilization and molecular characterization of active galanin receptors from rat brain.Biochemistry31, 2415–2422 (1992).

    Article CAS  Google Scholar 

  19. White, J.H. et al. Heterodimerization is required for the formation of a functional GABA(B) receptor.Nature396, 679–682 (1998).

    Article CAS  Google Scholar 

  20. Jones, K.A. et al. GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2.Nature396, 674–679 (1998).

    Article CAS  Google Scholar 

  21. Canals, M. et al. Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer.J. Neurochem.88, 726–734 (2004).

    Article CAS  Google Scholar 

  22. Canals, M. et al. Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer.J. Biol. Chem.278, 46741–46749 (2003).

    Article CAS  Google Scholar 

  23. Percherancier, Y. et al. Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers.J. Biol. Chem.280, 9895–9903 (2005).

    Article CAS  Google Scholar 

  24. Breit, A., Lagace, M. & Bouvier, M. Hetero-oligomerization between beta2- and beta3-adrenergic receptors generates a beta-adrenergic signaling unit with distinct functional properties.J. Biol. Chem.279, 28756–28765 (2004).

    Article CAS  Google Scholar 

  25. Uezono, Y. et al. Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence fromXenopus oocyte and baby hamster kidney cell expression system.Am. J. Physiol. Cell Physiol.290, C200–C207 (2006).

    Article CAS  Google Scholar 

  26. Saxton, M.J. Lateral diffusion in an archipelago. Distance dependence of the diffusion coefficient.Biophys. J.56, 615–622 (1989).

    Article CAS  Google Scholar 

  27. Jun, J.E. & Goodnow, C.C. Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling.Nat. Immunol.4, 1057–1064 (2003).

    Article CAS  Google Scholar 

  28. Davis, S.J. & van der Merwe, P.A. The structure and ligand interactions of CD2: implications for T-cell function.Immunol. Today17, 177–187 (1996).

    Article CAS  Google Scholar 

  29. Davis, D.M. & Dustin, M.L. What is the importance of the immunological synapse?Trends Immunol.25, 323–327 (2004).

    Article CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Wise (GlaxoSmithKline) for the gift of the GABAβR2 template, and E. Evans (Nuffield Dept. Clinical Medicine, Oxford University) and J. McIlhinney (MRC Anatomical Neuropharmacology Unit, Oxford University) for helpful discussion. This work was supported by the Wellcome Trust, the Rhodes Trust and the Programa Operacional Ciência e Inovação 2010, cofunded by the European Regional Development Fund.

Author information

Authors and Affiliations

  1. Nuffield Department of Clinical Medicine and Medical Research Council, Human Immunology Unit, Weatherall Institute of Molecular Medicine, The University of Oxford, Oxford Radcliffe Hospital, Oxford, OX3 9DU, UK

    John R James, Andrea Iaboni & Simon J Davis

  2. Group of Cell Activation and Gene Expression, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, Porto, 4150-180, Portugal

    Marta I Oliveira & Alexandre M Carmo

  3. Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar, 2, Porto, 4099-003, Portugal

    Marta I Oliveira & Alexandre M Carmo

Authors
  1. John R James

    You can also search for this author inPubMed Google Scholar

  2. Marta I Oliveira

    You can also search for this author inPubMed Google Scholar

  3. Alexandre M Carmo

    You can also search for this author inPubMed Google Scholar

  4. Andrea Iaboni

    You can also search for this author inPubMed Google Scholar

  5. Simon J Davis

    You can also search for this author inPubMed Google Scholar

Contributions

J.R.J., M.I.O. and A.I. executed the experiments; J.R.J., A.M.C. and S.J.D. formulated the experiments and wrote the manuscript.

Corresponding author

Correspondence toSimon J Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Residual analysis of BRET data for all constructs used. (PDF 287 kb)

Supplementary Table 1

Numerical representation of goodness-of-fits. (PDF 27 kb)

Supplementary Note

Why the dependence of transfer efficiency (BRETeff) on acceptor/donor ratio is systematically different for randomly interacting proteins versus oligomeric proteins, without recourse to theory. (PDF 65 kb)

Rights and permissions

About this article

Cite this article

James, J., Oliveira, M., Carmo, A.et al. A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer.Nat Methods3, 1001–1006 (2006). https://doi.org/10.1038/nmeth978

Download citation

This article is cited by

Access through your institution
Buy or subscribe

Associated content

G protein—coupled receptors: too many dimers?

  • Martin J Lohse
Nature MethodsNews & Views

Advertisement

Search

Advanced search

Quick links

Nature Briefing

Sign up for theNature 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
[8]ページ先頭
©2009-2025 Movatter.jp