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.

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIBbinding to the cell surface

“…Although GPCR oligomerization is reported, there are few examples of complexes that include more than two receptor proteins; one is that of the cannabinoid CB 1 /dopamine D 2 / adenosine A 2A receptor oligomers identified by SRET (25). Using two energy transfer approaches, BRET-BiFC and SRET, we identified heterocomplexes formed by two members of the GPCR family (CXCR4 and CCR5) and one of the Ig superfamily (CD4).…”

“…To confirm heterotrimerization, we used a sequential BRET/ FRET technique (SRET) (25). We transiently cotransfected 293T cells with a constant amount of CD4-Rluc (BRET donor) and CXCR4-CFP (BRET acceptor and FRET donor), and increasing amounts of CCR5-YFP (FRET acceptor); the SRET signal was positive and saturable (SRET max 197.1 ± 23.19, SRET 50 18.53 ± 7.74) (Fig.…”

“…3A), but not the total number of CXCR4 complexes (FRET max ). FRET 50 reflects the apparent affinity of a given interaction (25,26), and its variation can indicate conformational changes in the complex partners, which translate into longer or shorter donor-acceptor distances and/or changes in their relative orientation. Our results are nonetheless also compatible with CCR5-mediated interference with CXCR4 homodimers.…”

Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors

“…The spectral signatures of the different receptors fused to either GFP 2 or YFP did not vary significantly from the determined spectral signatures of the fluorescent proteins alone. For protein-YFP expression quantification, linear unmixing was done taking into account the spectral signature as described by Zimmermann et al (28,29) to separate the two emission spectra; the sample fluorescence is the fluorescence calculated as described minus the fluorescence of cells expressing only protein-Rluc and protein-GFP 2 . Second, we performed quantification of proteinRluc expression by determination of the luminescence due to protein-Rluc.…”

Design, synthesis and biological evaluation of bivalent ligands against A1–D1 receptor heteromers

“…Over the last 30 years, many researchers have identified that GPCRs can form dimeric and higher-order oligomers in natural tissues [1,2] . In comparison with the constituting receptors, GPCR heteromers, which have different characteristics, are important for the understanding of receptor function and pharmacology.…”