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Detection of heteromerization of more than two proteins by sequential BRET-FRET

Carriba, Paulina, Navarro, Gemma, Ciruela, Francisco, Ferré, Sergi, Casadó, Vicent, Agnati, Luigi, Cortés, Antoni, Mallol, Josefa, Fuxe, Kjell, Canela, Enric I, Lluís, Carmen and Franco, Rafael 2008. Detection of heteromerization of more than two proteins by sequential BRET-FRET. Nature Methods 5 (8) , pp. 727-733. 10.1038/nmeth.1229

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Subjects: Q Science > QR Microbiology
Publisher: Nature Publishing Group
ISSN: 1548-7091
Last Modified: 17 Mar 2021 02:42

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