Sensing conformational changes in metabotropic glutamate receptors.

G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are targets of more than 25% of the medications currently on the market. Metabotropic glutamate receptors (mGluRs), family C GPCRs, modulate synaptic transmission and neuronal excitability throughout the central nervous system and thus are promising targets for the treatment of various neurological and psychiatric disorders (1). Understanding the detailed activation mechanisms of mGluRs is therefore crucial to the development of new therapeutics. Although extensively studied over the last decade, the conformational changes involved in receptor activation are still a matter of debate (2). In PNAS, Doumazane et al. (3) describe how they have developed an innovative FRET-based approach to monitor these conformational changes upon ligand binding. Their assay not only helps to resolve controversy regarding the conformational changes involved in receptor activation but also facilitates our understanding of the mode of action of allosteric modulators. This approach therefore provides a powerful tool for drug screening. Family C GPCRs are obligate dimers; in the case of mGluRs, the two protomers are covalently connected via a disulfide bond (2, 4, 5). Each protomer contains a large extracellular domain (ECD) responsible for orthosteric ligand binding. The ECD is composed of a Venus flytrap (VFT) bilobate domain containing the ligand-binding site and a cysteine-rich domain that connects the VFT to the seven-helix transmembrane domain (TMD) (for review, see ref. 2) (Fig. 1). The binding of an agonist to the VFTs induces a series of conformational changes throughout the receptor, leading to the activation of intracellular effectors by the TMD and downstream signaling (2). How these domains work together to activate the coupled effectors remained poorly understood until 2000 and 2002, when the first crystal structures of the extracellular ligandbinding region of mGluR1 were solved (6, 7). The VFTs exist in two major states: an open state (o) and a closed state (c) (for review, see ref. 2). In the absence of agonist or in the presence of antagonist, both VFTs were thought to be open (oo), with the binding of one or two agonists inducing the closure of one (co) or two (cc) of the VFTs in the dimer, respectively. It has been proposed that the closure of at least one VFT induces a large change in the relative orientation of the two VFTs (2), which transitions the receptor dimer from a resting (R) to an active (A) orientation, thereby defining three major states, Roo, Aco, and Acc (2) (Fig. 1). In the putative R state, the distance between the two lobe 2s of the dimeric VFTs is greater than in the agonistbound A state. In contrast, the N-terminal lobe 1s are farther apart in the active state (Fig. 1). However, publication of the structures of dimeric VFTs in the closed conformation in the presence of an antagonist and in the open state in the presence of an agonist (8, 9) contradicted this model. Doumazane et al. (3) used a combination of innovative technologies to specifically label the extracellular N termini of cell surface mGluR2 receptors with fluorophores compatible with time-resolved FRET (tr-FRET) measurements, which read out on the distance between the two fluorophores (10). According to the model described above, the N termini move away from each other in the active state (Fig. 1), suggesting that receptor activation would be associated with a decrease of the FRET signal. Consistent with this hypothesis, glutamate, as well as four other agonists, decreased the FRET efficiency, reflecting the proposed agonist-stabilized closure of the VFTs and associated reorientation of the dimeric VFTs. Using various biochemical and pharmacological tools, as well as mutagenesis analysis, the authors were able to correlate these conformational changes with receptor activation. Importantly, Doumazane et al. obtained similar results with both mGluR1 andmGluR3, clarifying what appear to be general activation mechanisms of mGluRs and highlighting the limits of crystallographic studies using isolated ECDs for the study of conformational changes associated with receptor activation (2, 8, 9). The technology developed by Doumazane et al. (3) also allows for the identification of partial agonists. The application of two wellknown mGluR agonists only partially decreased the intramolecular FRET efficiency, consistent with their pharmacological characterization as partial agonists. However,