Elucidating the molecular logic of a metabotropic glutamate receptor heterodimer

Glutamate, the primary excitatory neurotransmitter in the mammalian central nervous system, acts on both ionotropic and metabotropic glutamate (mGlu) receptors—class C G protein-coupled receptors (GPCRs). mGlu receptors modulate neuronal excitability and synaptic transmission, making them promising therapeutic targets for various neuropsychiatric disorders, including Alzheimer's, Parkinson's, depression, stress-related disorders, and schizophrenia. There are eight mGlu receptor subtypes, categorized into three groups based on sequence similarity, G protein coupling, and pharmacology. Group I (mGlu₁ and mGlu₅) couples to Gq/11, while Groups II (mGlu₂ and mGlu₃) and III (mGlu₄, mGlu₆, mGlu₇, and mGlu₈) primarily couple to Gi/o/Go. mGlu receptors form stable dimers in the endoplasmic reticulum before trafficking to the plasma membrane. Each protomer consists of a large amino-terminal venus flytrap domain (VFD) containing the orthosteric glutamate-binding site. Agonist binding in the VFDs triggers structural changes propagated to the seven transmembrane domains (7TM), leading to G protein activation. Beyond homodimers, mGlu protomers can heterodimerize, significantly impacting receptor pharmacology. The study of mGlu receptor heterodimerization, particularly mGlu₂₄ heteromers found in the cortex and striatum, is crucial for understanding their physiological roles and developing precision medicine approaches for selective therapeutic targeting. Previous research on mGlu₂₄ heteromers, using electrophysiological studies and various mGlu₄ PAMs (e.g., VU0155041, Lu AF21934, PHCCC), revealed differential PAM activity at different synaptic locations, suggesting that the unique pharmacology of heterodimers versus homodimers might underlie these differences. However, traditional signaling assays cannot distinguish between homo- and heterodimers, hindering the understanding of their individual functions and pharmacological properties. To address this, the researchers developed a novel CODA-RET assay to investigate signaling from defined homodimers or heterodimers.

Existing research highlights the significant role of metabotropic glutamate receptors (mGluRs) in various neurological and psychiatric disorders. Studies have demonstrated the potential of mGluRs as therapeutic targets for conditions like Alzheimer's disease, Parkinson's disease, depression, and schizophrenia. The focus has increasingly shifted towards understanding the functional diversity arising from mGluR heterodimerization, as these complexes exhibit distinct pharmacological profiles compared to their homodimeric counterparts. Early work established the existence of mGluR heterodimers, and more recently, advanced techniques such as nanobody-based sensors provided direct evidence for endogenous mGlu₂₄ heteromers in various brain regions. Electrophysiological studies revealed differential responses to various mGlu₄ positive allosteric modulators (PAMs), indicating functional heterogeneity. However, a major challenge in studying mGluR heterodimers lies in the difficulty of isolating signaling from specific heteromeric complexes in the presence of homomeric populations. This limitation has motivated the development of innovative techniques like the complemented donor-acceptor resonance energy transfer (CODA-RET) assay employed in this study.

The researchers employed a novel complemented donor-acceptor resonance energy transfer (CODA-RET) assay to study the signaling of defined mGlu receptor homodimers and heterodimers. This assay utilizes a split *Renilla reniformis* luciferase 8 (RLuc8), divided into N-terminal (L1) and C-terminal (L2) fragments. These fragments are nonfunctional when separate but reconstitute a functional luciferase when brought into close proximity. By fusing these RLuc8 fragments to the C-termini of mGlu₂ and mGlu₄ protomers, the researchers could specifically control the complemented species and selectively measure G protein recruitment by defined homo- or heterodimers. mGlu homodimers formed by protomers fused to only L1 or L2 remain optically silent. Bioluminescence resonance energy transfer (BRET) between complemented RLuc8 (donor) and monomeric Venus (mVenus)-tagged Gα₀ subunit (acceptor) was used to monitor G protein recruitment upon receptor activation. The study further incorporated targeted mutagenesis of receptor protomers and computational docking to investigate the molecular mechanisms underlying the differential pharmacological activity observed. Multiple mGlu₄-specific PAMs were tested in CODA-RET assays with defined mGlu₄₄ homomers and mGlu₂₄ heteromers. Molecular docking, using the recent cryo-EM structure of the human mGlu₄₄ homodimer-G complex, was employed to predict PAM binding sites within the mGlu₄ 7TM domain. Mutagenesis experiments validated the docking predictions by investigating the effects of specific mGlu₄ mutations on PAM activity. Orthosteric agonist activation mechanisms (cis- and trans-activation) were explored using CODA-RET combined with mutations that block orthosteric agonist binding or G protein coupling. Finally, a BRET-based assay measuring cAMP inhibition was used to validate the CODA-RET findings at the level of downstream signaling.

The CODA-RET assays revealed that mGlu₄ PAMs binding to the upper allosteric pocket in the 7TM domain enhanced activation in both mGlu₄₄ homomers and mGlu₂₄ heteromers, whereas PAMs binding to the lower pocket were only effective in homomers. Molecular docking and mutagenesis experiments confirmed the distinct binding sites for these PAMs. Both protomers in mGlu₂₄ heteromers exhibited cis-activation by orthosteric agonists, signaling independently with no detectable trans-activation. However, upper pocket mGlu₄ PAMs enabled trans-activation from mGlu₄ to mGlu₂ and enhanced mGlu₂ cis-activation. In contrast, mGlu₂ PAMs only enhanced mGlu₂ cis-activation without facilitating trans-activation. Experiments using mutations to control which protomer binds agonists or G proteins confirmed that mGlu₄₄ homodimers can be both cis- and trans-activated by orthosteric agonists. The mGlu₂₄ heteromer exhibited only cis-activation of each protomer by its respective orthosteric agonist; however, upper pocket mGlu₄ PAMs enabled trans-activation from mGlu₄ to mGlu₂. This trans-activation was not observed with mGlu₂ PAMs or in the opposite direction (mGlu₂ to mGlu₄). A BRET-based cAMP inhibition assay corroborated these findings, demonstrating that upper pocket mGlu₄ PAMs enable trans-activation at the level of downstream signaling.

This study successfully addressed the hypothesis that the differential activity of mGlu₄ PAMs in vitro and in vivo could be explained by their distinct pharmacology at mGlu₄₄ homomers and mGlu₂₄ heteromers. The findings confirm that only upper pocket mGlu₄ PAMs are active at mGlu₂₄ heteromers. Molecular docking and mutagenesis experiments elucidated the distinct binding pockets and their role in differential PAM activity. The study demonstrates a complex interplay of cis- and trans-activation mechanisms in mGlu receptor homodimers and heteromers. The unique ability of upper pocket mGlu₄ PAMs to enable trans-activation highlights the functional asymmetry within mGlu₂₄ heteromers. The results contrast with previous studies, potentially due to methodological differences in receptor expression and assay systems. The findings provide valuable insights into the molecular logic of allosteric modulation in mGlu receptor heteromers, paving the way for the development of heteromer-selective compounds for precision therapeutics.

This research provides a comprehensive mechanistic understanding of mGlu₂₄ heterodimer activation and allosteric modulation. The discovery of distinct allosteric pockets and their differential coupling to activation in homo- and heteromers has important implications for drug discovery. The CODA-RET assay offers a powerful tool for studying signaling from defined dimeric receptors. Future research should focus on identifying small-molecule allosteric modulators that selectively target specific mGlu receptor heteromers, enabling the development of precision therapies for neuropsychiatric disorders.

The study primarily focuses on in vitro experiments using heterologous cell systems. The findings may not fully reflect the complexities of mGlu receptor function and interactions within the native brain environment. The specific binding modes and conformations of PAMs within the allosteric pockets require further investigation using advanced structural biology techniques. While the cAMP assay validates findings at the downstream signaling level, more in vivo studies are needed to confirm the translational relevance of these findings.