Nature Methods
- 5, 727 - 733 (2008)
Published online: 29 June 2008; | doi:10.1038/nmeth.1229
Detection of heteromerization of more than two proteins by sequential BRET-FRETPaulina Carriba1, 6, Gemma Navarro1, 6, Francisco Ciruela1, Sergi Ferré2, Vicent Casadó1, Luigi Agnati3, Antoni Cortés1, Josefa Mallol1, Kjell Fuxe4, Enric I Canela1, Carmen Lluís1 & Rafael Franco1, 51
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. 2
Behavioral Neuroscience Branch, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Department of Health and Human Services, 251 Bayview Blvd., Baltimore, Maryland 21224, USA. 3
Department of Biochemical Sciences, University of Modena, Via Campi 287, 41100 Modena, Italy. 4
Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 17177 Stockholm, Sweden. 5
Present address: Center for Applied Medical Research, Neurociencias, Avda. Pio XII 55, 31008 Pamplona. Spain. 6
These authors contributed equally to this work.
Correspondence should be addressed to Rafael Franco rfranco@unav.es 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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