Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

Etienne Doumazanea,b,c,1, Pauline Schollera,b,c,d,1, Ludovic Fabrea,b,c,d, Jurriaan M. Zwierd, Eric Trinquetd, Jean-Philippe Pina,b,c,2, and Philippe Rondarda,b,c,2 aCentre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France; bInstitut National de la Santé et de la Recherche Médicale U661, F-34000 Montpellier, France; cUniversités de Montpellier 1 and 2, F-34000 Montpellier, France; and dCisbio Bioassays, F-30200 Codolet, France

AUTHOR SUMMARY

Cell-surface membrane proteins made of a large extracellular play key roles in cell physiology domain that binds glutamate and cell–cell communication and and is associated with a seven- as such represent major thera- helix transmembrane domain peutic targets. Many of these that mediates G-protein acti- proteins serve as cell-surface vation (Fig. P1A). Agonists receptors, which are activated by (compounds that activate the extracellular messengers such as receptor, as glutamate does) light, small molecules, peptides, bind to a region within the ex- or proteins. Many cell-surface tracellular domain known as the membrane proteins are com- “Venus flytrap bilobate domain” posed of multiple domains and (VFT). The VFT oscillates be- polypeptide chains. Upon bind- tween two major states: an open ing of the messenger to the pro- state that is stabilized by antag- tein’s extracellular domain, onists (compounds that bind to structural changes in the trans- the receptor and prevent acti- membrane domain activate the vation) and a closed state that is receptor and lead to intracellular stabilized by agonists and is re- signaling events. quired for receptor activation. The neurotransmitter known However, it remains unclear as “glutamate” can activate two how VFT closure leads to types of receptors: ionotropic and transmembrane domain-mediated metabotropic. Ionotropic recep- activation of G protein, despite tors are glutamate-gated chan- the publication of crystal struc- nels responsible for fast excitatory tures showing the dimeric neurotransmission; metabotropic VFTs with bound agonists and glutamate receptors (mGluRs) antagonists (3). activate intracellular pathways In the present study, we ana- through heterotrimeric (i.e., con- lyzed the movement of subunits taining three different subunits) within mGluR dimers at the Gproteins. Fig. P1. Dynamics and allosteric properties of the SNAP-mGlu2 by time- level of the VFTs. In particular, mGluRs are part of the large resolved FRET. (A) The SNAP-tagged receptor labeled with fluorophores, we examined how these move- family of G protein-coupled compatible with the measurement of intramolecular FRET change: The ments are related to receptor receptors (GPCRs). These pro- high signal in the absence of ligand or in the presence of the competitive activation. To accomplish this antagonist LY341495 becomes lower in presence of glutamate or other goal, we used innovative tech- teins sense glutamate molecules agonists. (B) Intramolecular FRET signal of SNAP-mGlu2 at the surface of nologies that labeled cell-surface outside the cell and trigger in- living cells in the presence of a saturating concentration of glutamate proteins with molecules that tracellular pathways that help C and of competitive antagonist LY341495. ( ) This FRET signal depends on fluoresce and therefore can be regulate synaptic transmission the efficacy of the agonist’s glutamate and LY354740 and the partial studied using a technique known in the nervous system (1). As agonist DCG-IV. (D) PAM stabilizes the active conformation of the mGluRs such, the mGluRs represent in the presence of the agonist, and the NAM stabilizes the resting conformation. Accordingly, a saturating concentration of the NAM potential therapeutic targets for fi the treatment of several neuro- decreased the agonist ef cacy and slightly reduced agonist potency; Author contributions: E.D., P.S., L.F., J.-P.P., meanwhile, the PAM enhanced both agonist efficacy and potency. and P.R. designed research; E.D., P.S., and logic and psychiatric disorders, L.F. performed research; E.D., P.S., L.F., J.M.Z., such as anxiety, depression, E.T., J.-P.P., and P.R. analyzed data; and E.D., pain, schizophrenia, and Par- P.S., J.-P.P., and P.R. wrote the paper. kinson disease. More generally, the mGluRs are part of a group The authors declare no conflict of interest. of GPCRs known as “class C,” which also contains structurally This Direct Submission article had a prearranged editor. related receptors for sweet and umami taste, calcium, basic See Commentary on page 5742. amino acids, and the inhibitory neurotransmitter GABA (2). 1E.D. and P.S. contributed equally to this work. A better understanding of how changes in protein shape alter 2To whom correspondence may be addressed. E-mail: [email protected] or the function of mGluRs would enable the development of [email protected]. improved drugs to modulate their activities. The mGluRs are See full research article on page E1416 of www.pnas.org. strict dimers (i.e., are composed of two subunits). Each subunit is Cite this Author Summary as: PNAS 10.1073/pnas.1215615110.

5754–5755 | PNAS | April 9, 2013 | vol. 110 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1215615110 Downloaded by guest on September 27, 2021 as “fluorescence resonance energy transfer” (FRET) (4, 5). This assay, only those responsible for signaling can be detected using PNAS PLUS method measures FRET efficiency, which reflects the distance be- a cellular functional assay. Thus, our findings strongly suggest tween two fluorophores. We show that glutamate induces a large that the receptor–G protein complex alone, representing a small and rapid decrease in the FRET efficiency (Fig. P1B), indicating an fraction of the cell surface receptors, is responsible for the important movement of the VFTs upon receptor activation. We functional response. show that this movement is both necessary and sufficient for re- Our study reveals important information about the activation ceptor activation using two types of receptors carrying mutations in and allosteric properties of mGluRs in living cells and about how fi their extracellular domain. These ndings demonstrate that gluta- drugs regulate mGluRs activity. Our data reveal how agonist mate activates these prototypical dimeric receptors by allowing or binding in the VFTs causes a scissoring movement between the stabilizing a new orientation of the extracellular domains. VFTs that activates the G protein-activating transmembrane SEE COMMENTARY Our method of observing the VFT states directly through domain. This mechanism explains why mGluRs are dimers and FRET measurement could help researchers better understand provides opportunities to develop drugs capable of modulating how various drugs regulate mGluRs. We found that some ago- their activity. These findings certainly will create possibilities nists induce a smaller change in FRET compared with glutamate, for the development of innovative modulators. Eventually, we and we confirmed through functional studies that these mole- find that mGluRs couple to G proteins with properties similar to cules are partial agonists (agonists that induce partial receptor activation) (Fig. P1C). With regard to mGluRs, many synthetic those of other GPCRs, despite important structural differences molecules were found to either facilitate or limit the action of (e.g., dimeric organization and the presence of a large agonists. These compounds, called “positive” and “negative” extracellular domain). allosteric modulators (NAMs and PAMs, respectively) interact in Most importantly, our approach will drive the development of the transmembrane domain and are considered promising drugs cell-surface receptor biosensors compatible with high-throughput for the treatment of various brain diseases. screening, which has not yet been achieved with conventional How can such compounds, acting in the transmembrane do- FRET technologies. This approach might prove useful in drug main, regulate mGluR activity so precisely? Using the FRET development and could help researchers better understand the assay, we show that PAMs and NAMs allosterically control the function of mGluRs and other related receptors, as well as relative movement of the extracellular domains (Fig. P1D). membrane receptors in general. These findings reveal a clear interaction between the dimeric VFT and the transmembrane domains, with the conformation of 1. Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: Physiology, one influencing the conformation of the other. pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295–322. 2. Pin JP, Galvez T, Prézeau L (2003) Evolution, structure, and activation mechanism of GPCRs activate intracellular G proteins, which in turn in- Pharmacol Ther – fl family 3/C G-protein-coupled receptors. 98(3):325 354. uence the receptor conformation and stabilize the active state 3. Muto T, Tsuchiya D, Morikawa K, Jingami H (2007) Structures of the extracellular of the receptor. Our findings confirm that this activation also regions of the group II/III metabotropic glutamate receptors. Proc Natl Acad Sci USA holds true for mGluRs, because the active state of VFTs is fur- 104(10):3759–3764. ther stabilized in the receptor–G protein complex. This obser- 4. Maurel D, et al. (2008) Cell-surface protein-protein interaction analysis with time- resolved FRET and snap-tag technologies: Application to GPCR oligomerization. Nat vation explains why the agonist affects the FRET signal at higher Methods 5(6):561–567.

concentrations than that required to generate a response in cells. 5. Doumazane E, et al. (2011) A new approach to analyze cell surface protein complexes PHARMACOLOGY Indeed, although all receptors can be detected in the FRET reveals specific heterodimeric metabotropic glutamate receptors. FASEB J 25(1):66–77.

Doumazane et al. PNAS | April 9, 2013 | vol. 110 | no. 15 | 5755 Downloaded by guest on September 27, 2021