Abstract
Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB1, dopamine D2 and adenosine A2A receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.
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Acknowledgements
This study was supported by grants from the Spanish Ministry of Education and Science (SAF2005-00170 and SAF2006-05481), the Fundació La Marató de Televisió de Catalunya (060110) and from Intramural Funds of the US National Institute on Drug Abuse.
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Carriba, P., Navarro, G., Ciruela, F. et al. Detection of heteromerization of more than two proteins by sequential BRET-FRET. Nat Methods 5, 727–733 (2008). https://doi.org/10.1038/nmeth.1229
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DOI: https://doi.org/10.1038/nmeth.1229
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