# Steering G protein activation by mGlu heterodimer.

Guillaume Lebon

► To cite this version:

Guillaume Lebon. Steering G protein activation by mGlu heterodimer.. Cell Research, 2023, 33 (10), pp.735-736. 10.1038/s41422-023-00843-x. hal-04153579

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Submitted on 14 Nov 2023

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# Steering G protein activation from mGlu heterodimer

Guillaume Lebon

Institut de Génomique Fonctionnelle, Univeristé de Montpellier, CNRS, INSERM.

Heterodimerisation of metabotropic glutamate receptors (mGluS) generates functional units that modulate the synapse activity, and displays strong therapeutic potential for treating brain disorders and psychiatry diseases. Here, Wang et al.,¹ solved the cryo-electron microscopy (cryo-EM) structures of mGlu₂-mGlu₃, and mGlu₂-mGlu₄ heterodimers, in various conformational states, revealing the role of each subunit in the asymmetric signalling of mGlu heterodimers, the molecular basis of their allosteric modulation and giving a rational to understand which subunit activates the G protein.

The metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that are broadly expressed in the central nervous system and play important roles in neurobiological function. There are eight subtypes of mGlu receptors that form homo- and heterodimers, the dimerisation being essential to their function. mGlu heterodimers were reported², including for mGlu₁-mGlu₅, mGlu₂-mGlu₄, mGlu₂-mGlu₃, mGlu₂-mGlu₇ mainly. The existence of mGlu₂-mGlu₄ heterodimers in mice brain was recently reported, highlighting their physiological importance and potential role in brain functions and disorders³,⁴. The mGlu heterodimers offer new opportunities for tuning the synaptic activity. Understanding the molecular basis of their dimerization and asymmetric activation mechanism has remained unresolved.

mGlu homodimers are activated by glutamate binding in the Venus flytrap domains stabilising the close state (c), and active conformation of the VFT dimer (A). These first events trigger the movement of the Cystein Rich domains (CDR) and conserved heptahelical transmembrane domains (7TM) promoting inter-subunit molecular contacts and stabilizing the active state of the receptor⁵⁻⁷. Oppositely, antagonist binding maintains the VFTs in an open state (o) and inactive (R) conformation of the dimer, preventing the reorganization of both subunits and inhibiting the receptor activation⁸. The twelve cryoEM structures reported in Wang et al. ¹illustrate the complexity and diversity of the mGlu₂-mGlu₃ and mGlu₂-mGlu₄ dimerization modes, positive allosteric modulation, signal transduction and the multiples conformation populating the mGlu heterodimer conformational landscape (Roo, Rco, Acc with or without G protein).

The structures of the mGlu₂-mGlu₃ heterodimer obtained in combination with the antagonist LY341495, the mGlu₂ selective Negative allosteric modulator (NAM) NAM563 and the mGlu₃ selective NAM LY2389575 represent diverse inactive states of mGlu₂-mGlu₃. All adopt a Roo conformation of the VFTs but with differences in the 7TM dimerization modes: mode I that is stabilized by cholesterol, mode II and III in which the 7TMs are closer but using different protein-protein interfaces.

mGlu₂-Glu₃ was also captured in a Rco intermediate inactive state, in dimerization mode I, with NAM563 bound to mGlu₂ 7TM but with an empty and open VFT, whereas mGlu₃ VFT is stabilized in close state by glutamate. The higher glutamate affinity of mGlu₃ is likely giving account for such intermediate inactive Rco state. A similar conformation is also reported in the presence of glutamate and mGlu₂ PAM JNJ-40411813, but with the difference that a glutamate molecule is bound to the mGlu₂ VFT that remains open. Together such
subtle differences in the Rco intermediate inactive state highlights part of the first sequential reorganization of the VFTs towards the active state of the receptor (Fig. 1).

The transition from intermediate inactive (Rco) to intermediate active state (Acc without G protein) of the mGlu₂-mGlu₃ and mGlu₂-mGlu₄ heterodimers brings the CRDs in close proximity and the reorganization of the 7TM dimerization mode, as previously described for mGlu homodimer. However, the 7TM interactions are shifted to an asymmetrical interface with some differences between the two heterodimers. The mGlu₃ subunit is tilted away from the Glu₂ subunit whereas the mGlu₄ is rotating anticlockwise by 20 degrees. By combining a mutation that blocks the G protein binding (F765S) and mutations that prevent the binding of glutamate (YADA), the functional analysis provides the final demonstration that the closure of mGlu₃ is likely sufficient for activating the G protein via mGlu₂ 7TM, in a trans-activation mechanism similar to the signal transduction mechanism of the heterodimeric GABAB receptor, a member of the class C GPCRs⁹.

The next question is what drives G protein activation by one protomer over the other in mGlu heterodimers? Wang et al. report the structures of mGlu₂-mGlu₃ in complex with mGlu₂ PAM JNJ40411813 and with the G protein and the mGlu₂-mGlu₄ in complex with JNJ40411813 and 88178 mGlu₄ PAM. While in the case of the mGlu₂-mGlu₄ heterodimer, mGlu₄ was previously identified as the subunit coupling to the G protein¹⁰, the G protein is now bound to the mGlu₂ subunit, highlighting the ability of mGlu₂ to activate the G protein in the heterodimer. The main parameter driving the identity of the subunit recruiting the G protein is suggested to be the respective stability of the 7TM inactive state, the less stable favoring G protein binding and activation. In that respect the conserved tryptophan W782⁶,⁵⁰ in TM6 happens to be central in this mechanism and for controlling the equilibrium between inactive and active state of the helical bundle. The movement of W⁶,⁵⁰, homologous position of the so-called toggle switch in class A GPCRs, is more constrained in mGlu₂ (W773⁶,⁵⁰), due to a hydrogen-bond established with N735⁵,⁴⁷. In mGlu₄ however, the position 5.47 (S760) is occupied by a serine that cannot form hydrogen-bond W798⁶,⁵⁰ making mGlu₄ more prone for activating the G protein. A similar but reverse situation takes place at the mGlu₂-mGlu₃ heterodimer where position 5.47 in mGlu₃ is an aspartate that is better H-bond accepter compared to mGlu₂ asparagine, making mGlu₃ 7TM inactive state more stable and explaining the drive towards mGlu₂ helical bundle for activating the G protein.

It is however possible to pharmacologically modulate G protein coupling in mGlu heterodimers by destabilizing the mGlu₂ inactive state by using a PAM switching the G protein activation from mGlu₄ to mGlu₂, or contrarily by increasing the stability of mGlu₂ 7TM inactive state using the selective mGlu₂ NAM563 and partially recovering some G protein activation from mGlu₃ 7TM. Moreover, allosteric modulators may also act on the ternary structure of the complex as illustrated by the mGlu₄ PAM ADX88178 that unexpectedly targets the interface between mGlu₂ and mGlu₄ 7TM, interacting with both subunits and triggers G protein activation through mGlu₄ only. Overall, it becomes then very clear that the allosteric modulation can be a powerful strategy for taking control of a given mGlu subunit and to orientate the signal outcome of the heterodimer. GPCR function can be twisted by molecules that exploits unusual large diversity of binding sites.

The study by Wang et al. is a great example of the multiple conformation, Roo, Rco, Acc (G protein free), Acc-G protein bound states, that sequentially leads to G protein activation of the mGlu heterodimers (Fig. 1). The activation mechanism of the mGlu₂-mGlu₄ and mGlu₂-mGlu₃ heterodimers open great opportunities for subunit selectivity, providing unique but specific signal transduction signature of a given heterodimer.
1. Wang, X. et al. Structural insights into dimerization and activation of the mGlu2–mGlu3 and mGlu2–mGlu4 heterodimers. *Cell Res* 1–13 (2023) doi:10.1038/s41422-023-00830-2.

2. Doumazane, E. et al. A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors. *FASEB J* 25, 66–77 (2011).

3. Xiang, Z. et al. Input-specific regulation of glutamatergic synaptic transmission in the medial prefrontal cortex by mGlu2/mGlu4 receptor heterodimers. *Sci Signal* 14, eabd2319 (2021).

4. Meng, J. et al. Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain. *Nat Chem Biol* 18, 894–903 (2022).

5. Seven, A. B. et al. G-protein activation by a metabotropic glutamate receptor. *Nature* 595, 450–454 (2021).

6. Lin, S. et al. Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4. *Nature* 594, 583–588 (2021).

7. Koehl, A. et al. Structural insights into the activation of metabotropic glutamate receptors. *Nature* 566, 79–84 (2019).

8. Nasrallah, C. et al. Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations. *Cell Rep* 36, 109648 (2021).

9. Shen, C. et al. Structural basis of GABAB receptor–Gi protein coupling. *Nature* 594, 594–598 (2021).

10. Liu, J. et al. Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer. *Elife* 6, e26985 (2017).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic model of mGlu heterodimer G protein activation and its allosteric modulation. Inactive state Roo displays some diversity with different dimerization modes (mode I, II, III). Glutamate binding induces the closure of one VFT that is likely driving the rearrangement of the heterodimer quaternary structure (Rco state). In the active state structure (Acc), and in the presence of G protein (Acc-G protein), both VFTs bind glutamate and are in closed state but with some differences at the helical bundle interface. The selection of the mGlu subunit that activates the G protein is dependent of the respective stability of the inactive state of each subunit and can be pharmacologically modulated by acting either in one of the 7TM domain (blue) or at their molecular interface (green).</figcaption></figure>