1. Introduction
Because they play a key role in cell communication, G protein-coupled receptors (GPCRs) constitute a very active area of research and represent the largest group of potential drug targets for medicinal chemistry.
During the past decades, research on GPCRs has taken advantage of several technical advances, the energy transfer-based techniques (ETTs) being one of the most fruitful. These techniques allow the detection of protein-protein interactions and their dynamic variation at “physiological” expression levels in living cells.
ETTs are based on the nonradiative transfer of energy between a donor and an acceptor molecule. Because the efficacy of energy transfer (ET) varies inversely with the 6th power of the distance between the donor and the acceptor, ET from the donor will result in the emission of light by the acceptor only if the two molecules are in close proximity (10-100A). The detection of ET between proteins fused to energy donor and acceptor, respectively, is therefore a reflection of molecular interaction between the proteins of interest. A second important parameter, which influences the amount of ET to the acceptor, is its relative orientation to the donor. Accordingly, a variation of ET can also reflect conformational changes within the molecule, to which the donor or the acceptor is appended. Several variants of ET approaches have been used successfully to study GPCR biology. Fluorescence Resonance Energy Transfer (FRET) is the method of choice when the principal aim of the study is to identify the subcellular compartment in which the interaction between two proteins occurs. FRET, between an excited fluorescent donor and a fluorescent acceptor, can be measured with several methods, which have been recently reviewed elsewhere (Eidne et al., 2002). Bioluminescence Resonance Energy Transfer (BRET) occurring between luciferase, in the presence of its substrate, and a fluorescent protein as acceptor is particularly well adapted for high-throughput tests (Boute et al., 2002).
2. ETTs to probe GPCR self-association and conformational changes induced by agonists
One of the most striking recent conceptual changes in the field of GPCRs has been the demonstration that these receptors, classically considered to function as monomers, are actually organized as homo- or hetero-oligomers (Table 1) (reviewed in Breitwieser, 2004 and Kroeger et al., 2003). Although the precise biological signification of the phenomenon is still a matter of debate, ETTs certainly played a major role in establishing that GPCR oligomerization is constitutive in living cells and that it occurs early in the biosynthetic pathway at “physiological” concentrations of receptors (Ayoub et al., 2002; Issafras et al., 2002; Terrillon et al., 2003). ETTs were also used to identify molecular domains involved in the oligomerization process in intact cells (Overton et al., 2003; Salahpour et al., 2004) and data obtained with ETTs contributed significantly to a model, which postulates that GPCR homo-oligomerization may be a prerequisite for their targeting from the endoplasmic reticulum to the plasma membrane (Salahpour et al., 2004).
A large number of studies have investigated the role of agonist activation on GPCR oligomer formation using ETTs. In most cases, GPCR activation did not result in significant changes of ET, consistent with the notion that constitutive oligomerization is independent of the activation state of the receptor (Ayoub et al., 2002). However, in apparent contradiction with the hypothesis above, the activation of some receptors was clearly associated with marked changes of ET (Eidne et al., 2002). Although some authors have interpreted these changes as evidence for agonist modulation of dimerization, studies addressing specifically this issue have concluded that agonist-dependent changes in ET most likely reflect internal conformational changes of the receptors (Ayoub et al., 2004). The possibility to observe ET changes after agonist challenge may depend on both the amplitude of conformational changes induced by the agonist in a given receptor and on the design of the ET experiment itself.
Conformational changes occurring upon receptor activation were directly probed by FRET experiments in which the donor and the acceptor were inserted at different positions within a single receptor protomer (Vilardaga et al., 2003). Different activation and deactivation kinetics of various GPCR (from millisecond to second) were measured in the presence of their agonists or antagonists, and correlations could be established between the timing of the hormonal effects elicited by the receptors and the rapidity of their conformational switch.
3. ETTs to probe signaling events downstream of GPCR activation
During the past several years, ETTs in living cells have challenged some aspects of one of the most solid models in biochemistry, the mechanism of heterotrimeric interpreted as evidence for rapid dissociation of the subunits. Surprisingly, however, during continuous stimulation the Gs protein remained in its activated state despite the desensitization of the physiological response, suggesting that whether or not receptors are phosphorylated they catalyze the G protein cycle (Janetopoulos et al., 2001). A very similar strategy was followed by a different group, who studied Gi activation by GPCRs in mammalian cells. However, FRET changes were dependent on the specific position of the FRET acceptor on the Gy subunit, both agonist-promoted increase and decrease in the signal being observed. This finding led the authors to suggest that the changes in FRET reflected conformational rearrangements of the heterotrimer rather than a true dissociation of the subunits (Bunemann et al., 2003). The detection of a persistent G protein heterotrimer during activation clearly questions our understanding of how G proteins propagate signals through either their Ga or their Gfty subunits.
Table 1 Energy transfer-based techniques to study GPCR biology: Key references
| Biological phenomenon | References | Receptor | Major findings |
| GPCR dimerization | Overton et al. (2000) | Yeast pheromone receptor | Constitutive GPCR
oligomerization shown by FRET |
| Rocheville (2000) | Somatostatin and dopamine receptors | GPCR oligomerization shown by agonist-induced change of FRET | |
| Angers et al. (2000) | i2-adrenergic receptor | Constitutive GPCR
oligomerization shown by BRET |
|
| McVey et al. (2001) | f 2-adrenergic and 5-opioid receptors | Monitoring receptor oligomerization by time-resolved fluorescence resonance energy transfer | |
| Mercier et al. (2002) | f 1- and f 2-adrenergic receptor | Self-association affinity of GPCR protomers measured by BRET | |
| Identification of dimerization domains | Overton and Blumer (2002) | Yeast pheromone receptor | Characterization of the receptor dimerizaiton interface |
| Overton et al. (2003) | Yeast pheromone receptor | Motifs involved in receptor dimerization | |
| Salahpour et al. (2004) | f 2-adrenergic receptor | TM6 participates in the receptor dimerization interface | |
| Detection of agonist-induced conformation changes | Vilardaga et al. (2003) | Parathyroid hormone and a2-adrenergic receptors | Rapid agonist-induced conformational changes monitored by intramolecular FRET |
| Ayoub et al. (2002) | MT2 and MT1/2 melatonin receptors | Ligand-induced changes of BRET within preexisting dimers | |
| Monitoring of ligand binding to GPCRs | Turcatti et al. (1996) | Tachykinin NK2 receptor | FRET between
fluorescence-labeled GPCRs and fluorescent agonists |
| Palanche et al. (2001) | Tachykinin NK2 receptor | Binding kinetics of a fluorescent agonist to a GFP-tagged GPCR, monitored by FRET | |
| Ayoub et al. (2004) | MT2 and MT1/2 melatonin receptors | EC50 values for ligand-induced changes of BRET correlate with ligand affinities | |
| G protein activation | Janetopoulos et al. (2001) Bunemann et al. (2003) | Dictyostelium G protein Mammalian Gi protein | Monitoring of Gs protein
activation by FRET Monitoring of Gi protein activation kinetics |
| f-arrestin recruitment | Angers et al. (2000) | f 2-adrenergic receptor and f -arrestin | Agonist-induced translocation of f-arrestin to GPCRs |
G protein activation. Receptor-mediated activation of G proteins was visualized in Dictyostelium discoideum by monitoring FRET between the Gas and the Gy subunits fused to energy donor and acceptor fluorescent proteins. According to the established model, receptor stimulation led to a decrease of FRET signal that was
ETTs are particularly well adapted to monitor the translocation dynamics of signaling and/or regulatory proteins to activated receptors. For example, the translocation of 6-arrestins to GPCRs has been monitored by BRET (Angers et al., 2000), and was proposed as a tool for the identification of orphan receptor ligands (Bertrand et al., 2002). In addition, although the ET-based monitoring of 6-arrestin translocation confirmed a concept that was previously delineated with other approaches, it constitutes a proof of concept of the potential interest of ETTs to investigate the dynamics of protein complex formation throughout GPCR-dependent signaling pathways.
