title: BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues.
abstract: The concept of oligomerization of G protein-coupled receptor (GPCR) opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still controversial. Demonstrating direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing this precision. Resonance energy transfer (RET) strategies are actually the most convenient ones. During the last two decades, bioluminescent resonance energy transfer and time-resolved fluorescence resonance energy transfer (TR-FRET) have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently, a fluorescent ligand-based GPCR labeling strategy combined to a TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues. Although the RET-based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. The advantages and the limits of different approaches will be reviewed and the consequent perspectives discussed.
authors: Institut de Génomique Fonctionnelle CNRS, UMR 5203, Montpellier, France.
authors: Martin
authors: M
authors: Cottet
authors: Orestis
authors: O
authors: Faklaris
authors: Damien
authors: D
authors: Maurel
authors: Pauline
authors: P
authors: Scholler
authors: Etienne
authors: E
authors: Doumazane
authors: Eric
authors: E
authors: Trinquet
authors: Jean-Philippe
authors: JP
authors: Pin
authors: Thierry
authors: T
authors: Durroux