Detection of heteromerization of more than two proteins by sequential BRET-FRET.
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.
Carriba
Paulina
P
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.
Navarro
Gemma
G
Ciruela
Francisco
F
Ferré
Sergi
S
Casadó
Vicent
V
Agnati
Luigi
L
Cortés
Antoni
A
Mallol
Josefa
J
Fuxe
Kjell
K
Canela
Enric I
EI
Lluís
Carmen
C
Franco
Rafael
R
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
D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011956:Receptors, Cell Surface