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Analysis of receptor oligomerization by FRAP microscopy

Nature Methods volume 6, pages 225–230 (2009)Cite this article

Abstract

Here we describe an approach to investigate di- or oligomerization of transmembrane receptors in living cells with fluorescence recovery after photobleaching (FRAP). We immobilized a defined fraction of receptors with antibodies and then measured lateral mobility of the nonimmobilized fraction by FRAP. We validated this approach with CD86 and CD28 as monomeric and dimeric reference proteins, respectively. Di- or oligomerization of G protein–coupled receptors is strongly debated. We studied human β-adrenergic receptors as prototypical G protein–coupled receptors and found that β1-AR shows transient interactions whereas β2-AR can form stable oligomers. We propose that this FRAP method can be widely applied to study di- or oligomerization of cell-surface proteins.

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Figure 1: Immobilization of proteins by anti-YFP.
Figure 2: Validation of the dual-color FRAP approach using monomeric and dimeric control proteins.
Figure 3: Detection of di- or oligomerization of receptors by dual-color FRAP microscopy.
Figure 4: β2-AR homomeric interaction in cardiac myocytes.
Figure 5: Effects of expression ratios on FRAP of β1-AR and β2-AR.

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Acknowledgements

We thank N.A. Lambert (Medical College of Georgia) for methodical advice and him as well as U. Zabel (University of Wuerzburg), C. Krasel (University of Reading) and S.J. Davis (Oxford University) for providing cDNA constructs. This work was funded by the Deutsche Forschungsgemeinschaft (SFB 487 TPA1 to M.J.L. and TPA10 to M.B.).

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  1. University of Würzburg, Institute of Pharmacology and Toxicology, Versbacher Str. 9, Würzburg, 97078, Germany

    Sandra Dorsch, Karl-Norbert Klotz, Stefan Engelhardt, Martin J Lohse & Moritz Bünemann

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  1. Sandra Dorsch
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  2. Karl-Norbert Klotz
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  3. Stefan Engelhardt
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Contributions

S.D. performed and analyzed experiments; S.D. and M.B. designed experiments; K.-N.K. and M.J.L. contributed important ideas to the experiments; S.E. provided neonatal rat cardiac myocytes and some materials; S.D., M.B., K.-N.K. and M.J.L. wrote the manuscript.

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Correspondence to Moritz Bünemann.

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Dorsch, S., Klotz, KN., Engelhardt, S. et al. Analysis of receptor oligomerization by FRAP microscopy. Nat Methods 6, 225–230 (2009). https://doi.org/10.1038/nmeth.1304

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