title: Detection of heteromerization of more than two proteins by sequential BRET-FRET.
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 CB(1), dopamine D(2) and adenosine A(2A) 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.
authors.affiliation: Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain.
authors.forename: Paulina
authors.initials: P
authors.lastname: Carriba
authors.forename: Gemma
authors.initials: G
authors.lastname: Navarro
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
authors.forename: Sergi
authors.initials: S
authors.lastname: Ferré
authors.forename: Vicent
authors.initials: V
authors.lastname: Casadó
authors.forename: Luigi
authors.initials: L
authors.lastname: Agnati
authors.forename: Antoni
authors.initials: A
authors.lastname: Cortés
authors.forename: Josefa
authors.initials: J
authors.lastname: Mallol
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
authors.forename: Enric I
authors.initials: EI
authors.lastname: Canela
authors.forename: Carmen
authors.initials: C
authors.lastname: Lluís
authors.forename: Rafael
authors.initials: R
authors.lastname: Franco
mesh_terms: D002460:Cell Line; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry
chemical_list: D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011956:Receptors, Cell Surface