Allosteric activation of the glutamate receptor mGlu2 by the serotonin receptor 5-HT_2A

Domain: chemistry
Published: 2026-02-27
Editorial outcome: minor_revision

Abstract
G protein-coupled receptor (GPCR) dimers and higher-order oligomers are the subject of intense
debates. The complexes formed between two or more different GPCRs enable the cell to integrate
several signals. But the mechanisms of the allosteric interaction within these oligomers remain
unclear. Here, we use as a model the heteromer formed in the brain between two targets of anti-
psychotic drugs, the metabotropic glutamate receptor 2 (mGlu2) and the serotonin receptor 5-HT .
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Using molecular dynamics, a nanobody-based sensor and resonance energy transfer assays, we
demonstrate that the 5-HT receptor behaves as a positive allosteric modulator by stabilizing the
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active conformation of mGlu2. Using cysteine cross-linking experiments and mGlu2 sensors, we
reveal the molecular basis for this allosteric modulation of the mGlu2 receptor by 5-HT , an effect
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also mediated by other GPCRs. Our results thus provide insights into the allosteric control of a
GPCR activity via heteromerization with another receptor.

Introduction
G protein-coupled receptors (GPCRs) are the largest family of membrane proteins and are the
target of around 30% of marketed drugs1, 2. Upon activation by extracellular stimuli, they transduce
signals by coupling to heterotrimeric G protein proteins from different families3. Given that
individual cells express dozens of distinct GPCR subtypes, it raises the question of how the cells
integrate signals from such a diverse receptor repertoire. For over two decades, GPCR dimers and
higher-order oligomers were viewed as a possible way of signal integration (Fig. 1a)4-7, but this
subject raised intense debates8-10. Today, although such complexes are being considered, a more
accepted view is that different GPCRs can interact transiently11, 12, but the functional consequence
of such interaction remains more than elusive13-15. It is therefore important to understand how one
GPCR subtype regulates the function of another, as such knowledge may enable the development
of drugs that precisely tune GPCR signaling16.

The molecular mechanisms of allosteric interaction between GPCRs within these
oligomers, and in particular within heteromers formed of two different receptors6, are still unclear.
The molecular basis of direct interactions between GPCRs begins to be understood, thanks to
recent structural17-21 and biochemical13, 22 studies that have revealed possible dimerization
interfaces between receptors. Interestingly, homodimers can adopt an asymmetric interface in the
active state19, 20, consistent with the asymmetric functioning reported for many class A GPCR
dimers23-25, the most abundant receptors accounting for 90% of mammalian GPCRs. Class C
GPCRs19, 20, which classically form obligate dimers26, have also been studied in this regard. This
stands in contrast to class A receptors, for which the monomer has been demonstrated to be the
necessary and sufficient signaling unit27. In dimers and higher-order oligomers formed by class A
GPCRs, the seven transmembrane domains (7TMs) play a key role in stabilizing complexes, with
almost all TMs that could be involved in dimerization or oligomerization interfaces21. For dimeric
class C GPCRs, rearrangement of the dimerization interface during activation is well-characterized.
In the inactive state, the dimer interface is generally stabilized by interactions involving TM4 and
TM5 from each protomers22, 28, 29. A major reorientation of the dimer interface is observed during
agonist activation, where the two TM6s come into contact, a conserved conformation that enables
G protein coupling30. Recent structures of class C GPCRs also revealed the structural basis of the
asymmetry that occurs at the 7TM interface during G protein coupling19, 20. The rearrangement of
the 7TM interface for other GPCRs, such as class A receptors, is unclear, and some studies have
also proposed a relative repositioning of these transmembrane domains between the protomers31.

On the other hand, the molecular basis of allosteric interactions between two different
GPCRs in a heteromer remains poorly defined. Indeed, very few structures of complexes between
two different 7TMs have been solved, with the exception of the obligatory class C GABA
B
heterodimer28, 32, the sweet taste receptor33-35 and the recently discovered mGlu heterodimers29, 36,
37. Heterodimeric complexes formed between a class A GPCR and a class C GPCR are valuable
models for studying receptor heteromers, owing to both structural and physiological insights.
Firstly, the 7TM interfaces of several class C GPCR dimers in the inactive and active states has
recently been well characterized19, 20. Secondly, several heteromers formed between class C and
class A GPCRs have been shown to be physiologically relevant, including combinations between
several mGlu receptors and receptors of neurotransmitters (serotonin, dopamine or adenosine
receptors), including the mGlu2-5-HT 38 , mGlu5-D139 and mGlu5-A -D240 heteromers. Among
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them, the properties of the mGlu2-5-HT heteromer have been extensively studied, as such
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heterocomplex has been proposed as an important target for antipsychotics38, 41-46. Nevertheless,
the structural basis for the allosteric control of the mGlu2 receptor by 5-HT remains elusive.
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In this study, we used the mGlu2-5-HT receptor complex as a model to examine the
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molecular basis of the allosteric interaction between a class A and a class C GPCR. This receptor
complex was first reported over fifteen years ago, and subsequent in vivo studies have confirmed
the negative synergy of the mGlu2 and 5-HT receptors during schizophrenia and antipsychotic
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treatment42, 47. For example, the antipsychotic effects of clozapine on the 5-HT receptor require
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the expression of mGlu2 receptor48. Furthermore, the activation of the mGlu2 receptor has been
proposed to promote the anti-dyskinetic and anti-psychotic effects of 5-HT in the treatment of
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Parkinson’s disease49. Through molecular modelling, resonance energy assays and cysteine
crosslinking, we demonstrate that 5-HT receptor behaves as a positive allosteric modulator,
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stabilizing the active conformation of mGlu2. Strikingly, we also find that this allosteric activation
of mGlu2 is not specifically mediated by 5-HT receptors, but also mediated by many other class
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A GPCRs, including the several dopamine and serotonin receptors. Collectively, our results
provide insights into the allosteric control of a GPCR activity through heteromerization.

Results
Direct interaction between mGlu2 and 5-HT receptors at cell surface
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We first verified the interaction between mGlu2 and 5-HT receptors by time-resolved
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fluorescence resonance energy transfer (TR-FRET) saturation assays50. HEK-293 cells were co-
transfected with Snap-tagged mGlu2 (SmGlu2) and Halo-tag 5-HT (H5-HT ) for orthogonal
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labeling of two receptors with non-cell permeant fluorescent substrates specific of these tags
attached to the extracellular N-terminal of each receptor. To minimize the difference in size of the
N-terminal region between mGlu2 receptor, which has a large extracellular domain (563 residues),
and 5-HT , which has a short N-terminal sequence (67 residues), the Halo-tag (33 kDa) and Snap-
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tag (21 kDa) were fused to the 5-HT and mGlu2 receptors respectively51. We also optimized the
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labelling conditions to obtain a strong FRET signal (Supplementary Fig. 1). Saturation curve
analysis showed a strong FRET signal between SmGlu2 and H5-HT (Fig. 1b and Supplementary
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Fig. 2), albeit lower than the FRET signal measured between the SmGlu2 and HmGlu2 subunits,
which form a constitutive homodimer26, 52. A strong FRET signal was also observed between
SmGlu2 and the Halo-tagged dopamine D1 receptor HD1 (N-terminal region of 20 residues)
(Supplementary Fig. 3). In contrast, a weak FRET signal was obtained between SmGlu2 and the
Halo-tagged M2 muscarinic receptor (HM2, this negative control containing 18 amino-acids in its
N-terminal region; Fig. 1b), as well as sphingosine-1-phosphate receptor 1 (HS1P1R, 41 amino-
acids in its N-terminal region; Supplementary Fig. 3). Notably, the FRET signal intensity did not
correlate with the N-terminal length of the class A GPCRs tested. These data demonstrate a strong
propensity for mGlu2 to form a complex with 5-HT , consistent with previous studies38, 42, 53.
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3D model of the mGlu2-5-HT complex
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Structures of the full-length mGlu2 receptor in the inactive and active states20, 29 revealed the
relative rearrangement of the transmembrane domain interface in the homodimer upon agonist
activation. In the inactive state, stabilized by a competitive antagonist bound into the glutamate
binding site and a negative modulator bound into a transmembrane domain of the homodimer, the
transmembrane interface is composed of TM4 and TM320 (Fig. 2a-b). In the active state, the two
TM6s form the homodimer interface20, 29 (Fig. 2c-d). These structural data are consistent with the
model of 7TM rearrangement we previously proposed based on cysteine cross-linking experiments
of the mGlu2 receptor expressed at the surface of living cells22. In our previous work, we showed
that the mGlu2 receptor in its active state can form a homo-oligomer via the TM4-TM5 interface.
This finding suggested that the mGlu2 receptor can form higher-order complexes with other
membrane proteins across this interface.

To investigate the heteromeric interface, we generated a realistic and unbiased 3D model of
the active mGlu2 homodimer in complex with the 5-HT receptor. As our study was carried out
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before the structures of the two receptors were available, thus an initial homology model was built
for the active mGlu2 homodimer, where the two protomers interact via a symmetrical TM5-TM6
interface, and the inactive 5-HT receptor. In all-atom molecular dynamics (MD) simulations of
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the isolated mGlu2 homodimer, the TM5-TM6 interface between the mGlu2 protomers
progressively decreases its contact surface on the intracellular side and rotates towards a TM6-
TM6 interface, which is maintained until the end of the simulation (Fig. 2e-f). Subsequently, a
heterodimeric mGlu2-5-HT complex between mGlu2 homodimer and a 5-HT monomer was
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assembled by protein-protein docking, and an-unbiased molecular dynamics revealed a
symmetrical interface TM4-TM5 between the two receptors. During this simulation, we observed
that the conformation of mGlu2 promoters was stable in the mGlu2-5-HT complex, with RMSDs
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similar to those observed in the mGlu2 homodimer alone (average RMSD of 2.0 Å for the protomer
interacting with 5-HT and 1.8 Å for the other; Supplementary Fig. 4a). Similarly, the 5-HT
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maintained initial inactive conformation was also stable throughout our MD simulations, either in
the mGlu2-5-HT complex or in monomeric form (average RMSDs of 2.0 Å and 2.1 Å,
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respectively, Supplementary Fig. 4b). Consequently, this initial model of the mGlu2-5-HT
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complex was deemed to be an appropriate tool for studying inter- and intra-receptor functional
effects and for guiding the subsequent cross-linking experiments. More recently, we have further
validated this model through a new complex model built from experimental structures, the cryo-
EM structures of the mGlu2 homodimer20 and 5-HT receptor54 (see Materials and Methods). To
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confirm their consistency, we submitted both models, based on homology modeling and on
experimental structures, to the same protocol of coarse-grained molecular dynamics simulations
(CG MD; see Methods). This level of representation is well suited for comparing the organization
and persistence of pre-formed dimeric interfaces, as it allows access to longer effective simulation
timescales than would be feasible with all-atom models while retaining the key features of protein–
protein contacts. Because our analysis focused on the stability and similarity of existing interfaces
rather than on association/dissociation processes or detailed kinetics, standard coarse-grained MD
simulations were sufficient for this purpose, and no enhanced sampling methods were required. In
these CG trajectories, both models displayed the same general orientation of the protomers
(Supplementary Fig. 5a and 7e), with several common contacts at the interfaces
(Supplementary Fig. 5b and Fig. 7f). Moreover, the stability of the interfaces was preserved
across the CG MD trajectories (Supplementary Fig. 6d, 7d and 7e).

Cys cross-linking to characterize the mGlu2-5-HT complex
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We first validated this model of interaction between mGlu2 and 5-HT receptors with receptors
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expressed at the cell surface of HEK293 cells. First, we show that the mGlu2 agonist LY379268
increases the FRET signal between the two receptors when co-transfected (Fig. 3a and
Supplementary Fig. 8). This indicates that additional heteromers between these two receptors are
formed, although we cannot exclude that the increase in FRET signal results from a conformational
change between the two receptors (Supplementary Fig. 9a). In contrast, we verified that the
competitive antagonist LY341495 of mGlu2 did not alter the saturation curve (Supplementary
Fig. 9b). This is consistent with our model that only the mGlu2 receptor in the active conformation
can form a complex with the 5-HT receptor (Fig. 2e-f).
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Next, to further validate our model of the mGlu2-5-HT complex, we performed a
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cysteine disulfide crosslinking analysis. This technique probes proximity (< 8Å) between
engineered cysteine residues, enabling the detection of possible protein-protein interactions on the
cell surface. This approach has been successfully applied to study the dimeric 7TM interfaces of
several class C GPCRs, including the mGlu222 and calcium-sensing (CaS) receptor homodimers55,
the mGlu2-7 heterodimer29 and the GABA
B
receptor hetero-oligomer56. To detect the crosslinked
heterodimer in blots, Snap-tagged S5-HT and Halo-tagged HmGlu2 subunits were used to allow
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their specific labeling at the cell surface using non-cell permeant fluorescent substrates. This
allows direct fluorescence visualization of the receptors on blots, as previously reported22. Prior to
probing the heterodimeric interface, we first designed a mGlu2 mutant that stabilizes the active
state of mGlu2 in the absence of agonist. Based on our 3D model, the mGlu2Y6.58C mutation was
introduced into Halo-tagged wild-type mGlu2 (Fig. 3b and Supplementary Fig. 9c-d). This
construct, in which the active TM6-TM6 interface is stabilized by a disulfide bridge between the
two Cys6.58, was named “Active control” (mGlu2Ctrl) (Fig. 3b). We have shown that this mGlu2Ctrl
construct has a strong constitutive activity upon CuP treatment, which promotes disulfide
crosslinking, by measuring the constitutive accumulation of inositol phosphate-1 (IP ; 1
Supplementary Fig. 9e). To verify the presence of disulfide bridge crosslinking between the two
Cys6.58 in the mGlu2 homodimer in blot analysis, we used a construct in which the natural
intersubunit disulfide bridge through Cys121 was removed (Supplementary Fig. 9f), as it was
done previously22. A majority of mGlu2 dimers were observed after CuP treatment, an effect
reduced by an mGlu2 antagonist (Supplementary Fig. 9f). Overall, these results demonstrate that
Cys6.58 effectively stabilizes the active conformation of mGlu2 homodimer.
Next, to crosslink the HmGlu2Ctrl and S5-HT receptors (Fig. 3c-d), we introduced Cys at
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positions 5.42 and 5.41 in the HmGlu2Ctrl and S5-HT receptors, respectively, based on our 3D
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model (Supplementary Fig. 9c-d). We quantified on blots the S5-HT monomer (M), dimer (D)
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and S5-HT cross-linked to mGlu2 (mGlu2-5HT oligomers; O) thanks to the fluorescent Snap
2A 2A
substrates labelling. The efficiency of cross-linking between the two receptors induced by CuP
was quantified by the change of the ratio of the amounts for O/(M+D+O) before and after CuP
treatment. Interestingly, when co-expressing these two mutants, we obtained a CuP-induced high
molecular weight band (over 300 kDa) (Fig. 3e-f) that was suppressed by treatment with the
reducing agent DTT (Supplementary Fig. 10a-b). A similar band was obtained with wild-type
HmGlu2 containing a Cys in position 5.42, but the amount of cross-linked oligomer was clearly
increased in the presence of the mGlu2 agonist LY379268 (Fig. 3g-h). This shows the advantage
of using the HmGlu2Ctrl construct over wild-type HmGlu2 to study the heteromeric interface by
crosslinking since it does not require agonist treatment. Finally, the heteromeric composition of
this complex was confirmed by labeling the HmGlu2 and the S5-HT constructs with the
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fluorescent substrate Halo-Tag Alexa Fluor 660 and Snap-Surface Alexa Fluor 782, respectively
(Supplementary Fig. 10c-e). The high molecular weight band was visualized with both
fluorophores, confirming it contains both receptor subunits.
Our crosslinking analysis revealed that the heteromeric interface between HmGlu2 and
S5HT involves TM5 of both receptors, while a distinct disulfide bond between TM6 helices
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stabilizes the active conformation of the mGlu2 homodimer (Fig. 3d). Furthermore, our structural
model of the heterocomplex suggests a potential additional interaction interface involving the TM4
helices of both receptors (Fig. 2e-f). This possibility is supported by prior evidence implicating
TM4 in the oligomerization interface of active mGlu2 homodimers22. In contrast, the dimerization
interface of the 5-HT receptor is unclear57, and the experiments above indicate that only TM5 is
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involved (Fig. 3e-h). Therefore, we investigated the S5-HT homodimer interface by Cys
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crosslinking (Supplementary Fig. 11a), before further studying the mGlu2-5-HT interface.
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Symmetrical Cys crosslinking showed that TM4 of S5-HT was extensively crosslinked in the
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homodimer, in addition to TM5 and TM1 (Supplementary Fig. 11b-e). These results indicate that
5-HT can homodimerize across two possible symmetrical interfaces involving either TM4-TM5
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or TM1 (Supplementary Fig. 11d-e).
The above experiments indicate that TM4 of both mGlu2 and 5-HT receptors may be
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involved in their direct interactions. We then systematically mapped possible crosslinks involving
the TM4 and TM5 of these two receptors (Fig. 4a-b and Supplementary Fig. 12). Our data
showed that the greatest number of cross-links were obtained between TM5 of HmGlu2 and TM4
of S5-HT receptor (Fig. 4a-c and Supplementary Fig. 12). In contrast, no crosslinking was
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detected between mGlu2 and either TM1 or TM6 of the S5-HT receptor (Supplementary Fig.
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13a-b). These results show that TM4s and TM5s of mGlu2 and 5-HT receptors are primarily
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responsible for heteromer formation (Fig. 4d). This is highly consistent to previous findings that
Ala4.40, Ala4.44 and Ala4.48 located in mGlu2 TM4 are necessary for the mGlu2 to form a GPCR
complex with 5-HT receptor43, 46.
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In summary, our 3D models of the mGlu2-5-HT complex, together with crosslinking
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evidence, have strongly demonstrated that mGlu2 can interact directly with 5-HT to form a
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heteromer at the cell surface via their TM4-TM5 interface. Based on cross-linking results for the
5-HT homodimer, we cannot exclude that a 5-HT homodimer stabilized by TM1-TM1
2A 2A
interface is formed in the mGlu2-5-HT complex (Supplementary Fig. 14).
2A

5-HT stabilizes the mGlu2 receptor active state
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We used our recently developed nanobody-based FRET sensor58 to monitor the active
conformation of the mGlu2 receptor by detecting the active state of the extracellular glutamate
binding domain (Fig. 5a). This sensor is based on the measurement of a FRET signal between a
pair of fluorescent anti-mGlu2 nanobodies in the presence of LY379268 (Fig. 5a-b). The DN10
nanobody specifically binds the active conformation of the mGlu2 receptor favored by the agonist,
while the DN1 is not sensitive to the conformational state of the mGlu2 receptor58, 59. Interestingly,
the increase in transfected 5-HT receptor in the cell in the presence of a fixed amount of
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cotransfected mGlu2 receptor corresponded to an increase in FRET signal (Fig. 5b and
Supplementary Fig. 15), even in the absence of LY379268. This effect was prevented by the
mGlu2 antagonist (Fig. 5b and Supplementary Fig. 15), but was not influenced by 5-HT
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agonist or antagonist (Fig. 5c). Altogether, these data demonstrate that 5-HT receptor, either in
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its inactive or active conformation, stabilizes the active conformation of the mGlu2 receptor. To
rule out the possibility that the DN10 nanobody is responsible for this increase in FRET by
stabilization of the active mGlu2 receptor, we monitored the conformational rearrangement of the
mGlu2 extracellular binding domain using our Snap-tagged mGlu2 sensor59-61. Consistent with the
nanobody-based FRET sensor results, the increase in transfected 5-HT receptor in the presence
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of a fixed amount of cotransfected mGlu2 receptor decreased TR-FRET ratio of SmGlu2 dimers
(Supplementary Fig. 16a, b) and increased in the proportion of the mGlu2 receptors in an active
state (Supplementary Fig. 16a, c). This effect occurs at lower concentration of 5-HT than when
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measured with the nanobody based assay, a difference that can be explained by the different timing,
the nanobody based assay being measured after overnight incubation with the nanobodies. Of
importance, this effect was not observed with the M2 receptor used as a negative control
(Supplementary Fig. 16d, e), since it does not have a tendency to interact directly to the mGlu2
receptor.
This conclusion is further supported by the behavior of a 5-HT receptor mutant (R173P)
2A
located in the intracellular part of TM3. This mutant is deficient in G protein coupling (Fig. 6a),
but it can still stabilize the active state of the mGlu2 receptor, as observed by the increased of
FRET signal (Fig. 5d). Of note, this 5-HT R173P mutant corresponds to the mutation of the Arg
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residue of the DRY motif that is highly conserved in class A GPCRs62, 63. Although mutation R173
into proline has not been reported to our knowledge in the 5-HT
2A
receptor, mutation of this residue
in other class A GPCRs strongly impairs G protein coupling63. Here, we show that R173P mutant
was expressed at the cell surface similarly to the wild-type (Supplementary Fig. 17a).
Furthermore, this mutation prevents the transactivation of 5-HT receptor by the LY379268
2A
activated mGlu2 receptor (Supplementary Fig. 17b-e), as measured by the intracellular calcium
release induced by mGlu2 in the presence of co-transfected 5-HT receptor44.
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We then confirmed the stabilization of the active state of mGlu2 receptor by 5-HT by
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measuring the accumulation of inositol phosphate-1 (IP ), using a chimeric Gqi protein to couple
1 9
mGlu2 to phospholipase C (PLC)22. The 5-HT R173P mutant increases the basal activity of the
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co-expressed mGlu2 receptor (Fig. 6b), which can be blocked by the competitive antagonist
LY341495 or a negative allosteric modulator that binds to the mGlu2 transmembrane domain22
(Fig. 6c). This increase in basal activity is rather modest, but it is significant. This finding is
consistent with other data above showing that 5-HT receptors stabilize only a fraction of the
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mGlu2 receptors in an active state (Fig. 5b and Supplementary Fig. 15a). In addition, as expected
from a low fraction of the mGlu2 receptors interacting with the 5-HT R173P mutant, no
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significant increase of the potency of the mGlu2 agonist by co-expression of the 5-HT R173P
2A
mutant is observed (Supplementary Fig. 17f). Finally, a lower intracellular calcium mobilization
by the LY379268 activated mGlu2 receptor in presence of Gqi and the 5-HT R173P mutant is
9 2A
measured (Fig. 6d), and it is not related to the mGlu2 cell surface expression level
(Supplementary Fig. 17g). This observation supports the effect of this 5-HT mutant in
2A
increasing the basal activity of mGlu2 receptor by a direct interaction. Indeed, it is recognized that
a basal activation of PLC, such as that resulting from a GPCR constitutive activity, can partly
empty the intracellular calcium stores, leading to a decrease in the calcium signal induced by the
activation of a PLC-coupled GPCR.
Altogether, these data demonstrate that 5-HT
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acts as a positive allosteric modulator on
mGlu2 receptor activation even under basal conditions where only ambient glutamate may be
present64.

The mGlu2 receptor active state is stabilized by other class A GPCRs
In order to clarify which GPCRs could interact with mGlu2 receptor at the cell surface, we
performed a systematic analysis with 44 different class A GPCRs tagged at their N terminus with
a Snap-tag, as illustrated in Figure 7a. By using the mGlu2 nanobody-based FRET sensor, a
number of these GPCRs generated a high FRET signal between the two mGlu2 nanobodies that
selectively bind on the active mGlu2, when coexpressed with HA-tagged mGlu2 receptor,
including 5-HT , the dopamine D1 to D4 receptors, while others generated lower but significant
2A
signals, such as the 5-HT , the adrenergic  and the delta opioid receptors. All others did no
2B 1A
generate a signal significantly higher than the background, including the histamine H1 to H3
receptors and the muscarinic M2 to M4 receptors. This was not explained by differences in the
amount of the receptors at the cell surface (Supplementary Fig. 18a,b) and there is no link with
the N-terminal length of these class A GPCRs such as short N-terminus for D1 receptor made of
20 residues, until the long N-terminus of the cannabinoid CB1 receptor which is formed by 111
residues.
We found that the Gs-coupled D1 receptor, which has a strong tendency to stabilize the
active conformation of mGlu2 receptor (Fig. 7a), in an expression level dependent manner (Fig.
7b). Consistently with this stabilization, the D1 receptor increased the basal accumulation of IP
1
by the co-expressed mGlu2 receptor (Fig. 7c), an increase that was not observed when the D1
receptor was expressed alone. This basal activation of mGlu2 receptor by D1 receptor is also
supported by the lower calcium mobilization of the agonist-induced mGlu2 when D1 receptor is
coexpressed (Supplementary Fig. 18c) despite a similar expression of mGlu2 (Supplementary
Fig. 18d). As for 5-HT receptor, no significant increase of the potency of the mGlu2 agonist by
2A
co-expression of the D1 receptor is observed (Supplementary Fig. 18e), consistent with a low
fraction of the mGlu2 receptors interacting with the D1 receptor. Altogether, these data indicate
the effect of D1 receptor increases the constitutive activity of mGlu2 by a direct allosteric
interaction.
To further support the specific allosteric effect of both 5-HT and D1 receptors on mGlu2
2A
receptor, we used the Gi/o-coupled M2 receptor, one of the class A GPCRs that generated no
increase of FRET signal by the nanobody-based mGlu2 sensor (Fig. 7a). This is consistent with
the low FRET signal between the M2 and mGlu2 receptors shown above (Fig. 1b and
Supplementary Fig. 3) showing that the M2 receptor is less prone to interact with mGlu2 receptor
than 5-HT receptor. Increasing the M2 receptor versus mGlu2 receptor expression ratio did not
2A
result in an increase in the nanobody-based FRET sensor for mGlu2 activation (Fig. 7d).
Consistent with this absence of effect, a M2 mutant dead for the coupling to G protein (R121P also
mutated in the DRY motif; Supplementary Fig. 19a), did not increase the basal accumulation of
IP by the co-expressed mGlu2 receptor (Fig. 7e), despite its similar expression at the cell surface
1
compared to that of the wild-type M2 receptor (Supplementary Fig. 19b). Furthermore, this M2
R121P mutant did not alter the potency of the mGlu2 agonist in IP accumulation (Supplementary
1
Fig. 19c) and in the calcium responses (Supplementary Fig. 19d), despite a similar expression of
mGlu2 (Supplementary Fig. 19e).
Altogether, this demonstrates that mGlu2 receptor not only can be allosterically activated
by 5-HT receptor, but also by other class A GPCRs, even under basal conditions where only
2A
ambient glutamate may be present64.

Methods
Materials
LY379268, LY341495, MNI137, 5-HT (5-Hydroxytryptamine), Ketanserin tartrate were
purchased from Tocris Bioscience (Bristol, UK). Dichloro (1,10-phenanthroline) copper(II) (CuP)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Muscarine were purchased from
MedChemExpress (Shanghai, China). Lipofectamine 2000 was obtained from Thermo Fisher
Scientific (Carlsbad, CA, USA). Snap-Surface® Alexa Fluor® 782 was from New England
Biolabs. Halo-Tag® Alexa Fluor®660 and Coelenterazine-h were from Promega (Beijing) Biotech
Co. IP-One Gq kit, Snap-Green, Snap-Lumi4-Tb, Snap-Red, Halo-Green and Halo-Lumi4-Tb
were from Revvity Cisbio (Codolet, France), respectively.

Plasmids and transfection
The PRK5 plasmids encoding wild-type rat mGlu2, with HA and Snap or Flag and Halo inserted
just after the signal peptide were constructed as previously reported56. The plasmids encoding HA
and Snap or Flag and Halo tagged wild-type rat 5-HT were obtained in the same way. All the
2A
Cysteine mutants for mGlu2 and 5-HT together with G protein activation defective mutant 5-
2A
HT R173P and M2R121P were generated by site-directed mutagenesis using the QuikChange
2A
mutagenesis protocol (Agilent Technologies). All constructs were confirmed by DNA sequencing.
HEK293 cells (ATCC, CRL-1573, lot: 3449904) were cultured in DMEM (Gibco)
supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). For cross-linking
assay, HEK293 cells were transfected in 12 well plate with plasmids of interest by Lipofectamine
2000. For IP accumulation measurements, cells were transfected with the chimeric G-protein
1
Gqi9 and the glutamate transporter EAAC1 to enable mGlu2 receptor couple to the phospholipase
C pathway22.

Fluorescence resonance energy transfer (FRET)
30 000 HEK293 cells per well in black non-transparent 96-well plates are transfected with fixed
20 ng of SmGlu2 plasmid cDNA and increasing amounts of HmGlu2, H5-HT or HM2R plasmid
2A
cDNA with 0, 5, 10, 20, 40, 60 and 80 ng, and completed to a total amount of 100 ng of plasmid
DNA per well with the empty vector pRK5. 24 hours after transfection, HEK293 cells were labeled
with solution of 100 nM of Snap-Lumi4-Tb and 60 nM of Halo-Green in Tag-lite buffer at 37°C
for 1 h. Afterwards, cells were washed three times with FRET buffer (20 mM HEPES, 146 mM
NaCl, 4.2 mM KCl, 1 g/L glucose, 0.1% BSA, pH adjusted to 7.4), and drugs were added to
investigate effects of agonist LY379268 and antagonist LY341495. Fluorescence of Lumi4
(excitation at 340 nm, emission at 620 nm, 50-s delay, and 450-s integration time), Green (485
nm, 520 nm, 0 s, and 1000 s), and TR-FRET (340 nm, 520 nm, 50 s, and 450 s) were collected
on an Infinite F500 spectrofluorimeter (Tecan, Männedorf, Switzerland). The expression of Snap-
tagged and Halo-tagged receptors were determined by the fluorescence intensity of Snap-Lumi4-
Tb and Halo-Green, respectively50.

Cell surface quantification
The expression of HA-Snap tagged or Flag-Halo tagged constructs in cell surface were detected
by ELISA or fluorescent labeling56 as previously described.

Nanobody based TR-FRET assay
30 000 HEK293 cells per well in white non-transparent 96-well plates were transfected with fixed
20 ng of SmGlu2 plasmid cDNA and increasing amounts of HmGlu2 plasmid cDNA with 0, 5, 10,
20, 40, 60 and 80 ng, and completed to a total amount of 100 ng of plasmid DNA per well with the
empty vector pRK5. 24 h after transfection, HEK293 cells were treated with or without drugs and
then labeled with solution of 7 nM of DN1- Lumi4-Tb and 15 nM of DN10-d2 in Tag-lite buffer
at 25°C overnight58. Afterwards, cells were washed three times with Tag-lite buffer. FRET
measurements were performed using the PHERAstar FS (BMG Labtech, German). Calculate the
ratio of the acceptor and donor emission signals for each individual well. Ratio=Signal 665
nm/Signal 620 nm * 104.

TR-FRET based mGlu2 conformational assay
30 000 HEK293 cells per well in black non-transparent 96-well plates are transfected with 20 ng
of SmGlu2 plasmid cDNA and increasing amounts (0, 5, 10, 20, 40, 60 or 80 ng) of H5-HT or
2A
HM2 plasmid cDNA, and completed to a total amount of 100 ng of plasmid DNA per well with the
empty vector pRK5. 24 h after transfection, HEK293 cells were labeled with solution of 100 nM
of Snap-Lumi4-Tb and 60 nM of Snap-Green in Tag-lite buffer at 37°C for 1 h. Afterwards, cells
were washed three times with Tag-lite buffer, and drugs were added to investigate effects of agonist
LY379268 (10 M) and antagonist LY341495 (10 M). The TR-FRET measurements were
performed on a PHERAstar FS microplate reader with the following setup: after excitation with a
laser at 337 nm (40 flashes per well), the fluorescence was collected at 520 nm for a 50-μs reading
after a 50-μs delay after excitation (window 1) or for a 400-μs reading after a 1,200-μs delay
(window 2). The FRET signal was determined by dividing the signal measured in window 1 by
the signal measured in window 2. Besides, the fluorescence of Snap-Lumi4-Tb and Halo-Lumi4-
Tb (excitation at 340 nm, emission at 620 nm, 50-s delay, and 450-s integration time) were
collected on a PHERAstar FS (BMG Labtech) to determine the expression of Snap-tagged and
Halo-tagged receptors, respectively, in separate batches of the same transfected cells.

Inositol phosphate measurements
30 000 HEK293 cells per well in white non-transparent 96-well plates were transfected with 20 ng
SmGlu2 and 60 ng H5-HT R173P of plasmid cDNA, or with fixed 20 ng of HmGlu2 plasmid cDNA
2A
and increasing amounts of SD1 or SM2R121P plasmid cDNA with 0, 5, 10, 20, 40, 60 and 80 ng, and
completed to a total amount of 100 ng of plasmid DNA per well with the empty vector pRK5. IP
1
accumulation in HEK293 cells was measured in the PHERAstar FS (BMG Labtech, German) by
using the IP-One HTRF kit (Revvity CisBio) according to the manufacturer’s recommendations.
For cross-linking experiments, cells were treated with 1.5 mM CuP in cross-linking buffer for 10
min (as described above for blot experiments) before measuring.

Intracellular calcium release
The intracellular calcium release measurements were performed as previously described79. 30 000
HEK293 cells per well were in black transparent 96-well plates were transfected with plasmids
encoding the indicated receptors (mGlu2, 5-HT
2A
R173P, D1 and M2R173P) with chimeric protein
Gqi , and then incubated for 24 h. The cells were then preincubated for 1 h with Ca2+-sensitive
9
Fluo-4 (Life Technologies). The fluorescence signals (excitation at 485 nm and emission at 525
nm) were measured for 60 s and recorded using a Flexstation 3 microplate reader (Molecular
Devices, Sunnyvale, CA, USA). After the first 20 s, cells were treated with agonist and the Ca2+
signals were indicated as stimulated increase in fluorescence response. Concentration response
curves were fitted using Graph Pad Prism.

Cross-linking and fluorescent-labeled blot experiments
200 000 HEK293 cells per well, seeded in 12-well plates, were transfected with wild type or mutant
1.05 g S5-HT and 0.15 g HmGlu2. After 36 h transfection, adherent HEK293 cells plated in
2A
12-well plates were labeled with 100 nM Snap- Alexa Fluor 782 or 100 nM Halo- Alexa Fluor 660
in culture medium at 37°C for 2 h. Then, cells were incubated with drug (each at 10 M) or PBS
at 37°C for 30 min. Afterwards, cells were treated with cross-linking buffer (1.5 mM Cu(II)-(o-
phenanthroline), 1 mM CaCl , 5 mM Mg2+, 16.7 mM Tris, pH 8.0, 100 mM NaCl) at room
2
temperature for 20 min. After incubation with 10 mM N-ethylmaleimide at 4 °C for 15 min to stop
the cross-linking reaction, cells were lysed with buffer (containing 50 mM Tris (pH 7.4), 150 mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemented with complete phosphatase inhibitor
cocktail (Roche) at 4 °C for 1 h. After centrifugation at 12,000×g for 30 min at 4 °C, supernatants
were mixed with loading buffer at 37 °C for 10 min. In reducing conditions, samples were treated
with additional 100 mM DTT in loading buffer for 10 min. Equal amounts of proteins were
separated by 6% SDS-PAGE and then transferred to nitrocellulose membranes (Millipore).
Membranes were imaged on Odyssey CLx imager (LI-COR Bioscience, Lincoln, NE, USA) at
700 nm and 800 nm56. The density of bands was calculated by ImageJ software (Bethesda, MD,
USA).

Homology modeling and assembly of mGlu2-5-HT complex model
2A
To generate the 3D model of mGlu2-5-HT complex, wild-type human mGlu2 7TM and full-
2A
length 5-HT were homology modeled form the crystal structure of mGlu1 7TM (PDB: 4OR2)80
2A
and 5-HT (PDB: 4IB4)81 using Modeller v9.16, respectively82. Prior to modeling, co-crystallized
2B
ligand ergotamine and non-native fusion protein between TM5 and TM6 were removed. Secondary
structure of non-crystallized intracellular loop 3 (ICL3) of 5-HT (Tyr249 to Gln314) was
2B
consensus predicted with PsiPred83, PSSPred84 and Jpred85 and then added with Modeller82.
Sequence alignments between mGlu1:mGlu2 and 5-HT :5-HT were generated with
2B 2A
PROMALS-3D84 and manually curated to ensure all insertions/deletions occurred in loop regions
only. For both receptors, from each structural template, 50 different homology models were
generated and ranked by positional criteria and zDOPE score86. For all systems, complete GPCR
homology models were subsequently energy minimized in the AMBER14SB force-field87 using
Chimera v1.11 in vacuum conditions88.
The activated mGlu2 homodimer model was superimposed onto the respective monomers
of the homodimeric -opioid receptor crystal structure (PDB: 4DKL)43. This homodimer model
was optimized through protein-protein docking with ROSIE web-server85, with the best fitting
homodimer selected based on highest ROSETTA I_sc score89 and lowest RMSD from 1000
docking solutions. After independent all-atom molecular dynamics simulations, conformations of
5-HT monomer and active mGlu2 homodimer were used to model the mGlu2-5-HT
2A 2A
heteromeric complex by superimposing individual 5-HT and one protomer of mGlu2 dimer onto
2A
the homodimeric adenosine A
2A
receptor crystal structure (PDSB: 4EIY)90, which was deemed the
most appropriate crystal structure available at the time. Protein-protein docking between the two
receptors was then performed with the ROSIE web-server85 for identification of the best possible
arrangement. The final oligomeric model was energy minimized without restraints in Chimera
v1.11 with the AMBER14SB force-field to optimize protein–protein interactions.
To further validate our proposed model above, we built another model for the complex by
using cryo-EM structures of mGlu2 homodimer and 5-HT receptor published after the beginning
2A
of this study. We use the inactive conformation of 5-HT (PDB: 7WC8)54 and the active
2A
conformation of mGlu2 (PDB: 7MTR)20 where dimeric 7TM interface is TM6-TM6. For both
receptors, ligands and any non-protein atoms were removed, residues or loops missing from the
structures were added with Modeller, and mutations were reverted to wild-type in Chimera88. The
Venus flytrap domains (VFTs) of the mGlu2 protomers were removed, so that the model of the
complex involved only the transmembrane domains of both receptors. Indeed, the VFTs control
the relative position of the 7TMs from each subunit, but this is already established the 7TM dimer
used as template, and the VFTs are not expected to be in direct contact of the 5-HT receptor.
2A
Also, it is worth noting that G proteins were not present in the mGlu2 active structure used for the
modeling (PDB: 7MTR). Following the same strategy used to build the previous model, 5-HT
2A
and a protomer of mGlu2 were superimposed on a homodimer of the A receptor (PDB: 4EIY)
2A
to form a heteromer with a TM4/5-TM4/5 interface between these two receptors. This model of
the mGlu2-5-HT complex was then submitted to the "Refinement" tool on the Haddock
2A
webserver91, following the "Simulated Annealing with centroid restraints" protocol, using the
default parameters. The structure with the most favorable Haddock score was selected.
One difference between the two models is the position of TM4 of the 5-HT receptor. In
2A
homology model, this helix is located between TM5 of one mGlu2 protomer and TM6 of the other
protomer. While in the new model, 5-HT TM4 moves towards to the TM5 of mGlu2 protomer,
2A
which explains the higher number of frequent contacts between mGlu2 TM5 and 5-HT TM4
2A
observed in this case (Supplementary Fig. 7-8). This difference probably results from its
conformation in the homology model, which predicted a shorter helix than the one observed
experimentally. However, despite these differences and the inherent limitations of homology
modeling, there were still several frequent contacts between mGlu2 and 5-HT which were
2A
common between the two models, and among them many involved TM5 of mGlu2 and TM4 of 5-
HT
2A
. Therefore, the first model adequately represented the interfaces of the complex, and
captured contacts between the protomers that are in agreement with the experimental results.

All-atom molecular dynamics (MD) simulations and coarse-grained (CG) analysis
All-atom MD simulations of 5-HT , mGlu2 homodimer alone and mGlu2-5-HT complex model
2A 2A
were performed using the CHARMM36 force-field92 with ACEMD93 on specialized GPU-
computer hardware. All systems were equilibrated for 28 ns each (except mGlu2-5-HT hetero-
2A
complex model, which was equilibrated for 56 ns) at 300 K and 1 atm, with positional harmonic
restraints on protein heavy atoms progressively released over the first 8 ns (first 16 ns for mGlu2-
5-HT complex model) of equilibration and then continued without constraints. After
2A
equilibration, 5-HT monomer, mGlu2 homodimer alone and mGlu2-5-HT heteromer model
2A 2A
were subjected to unbiased continuous production runs under the same conditions for 5 µs each.
Analysis of 5-HT monomer, mGlu2 homodimer alone and mGlu2-5-HT heteromer model MD
2A 2A
simulations was performed using VMD software 1.9.294. The protomer conformational change of
each system was respectively measured by means of root mean square deviation (RMSD).
The two hetero-complex models, which are based on homology modeling and cryo-EM
structures respectively, were further analyzed through CG MD simulations set up in the
CHARMM-GUI web server95 and performed with GROMACS v.2019.696. The systems were
described by Martini 2.2 representation97, 98, with the elnedyn elastic network99 and inserted into a
POPC membrane. The elastic network connects the beads representing the backbone of the protein,
preventing conformational changes; therefore, the initial conformations of both receptors were
preserved throughout the trajectories. However, it is important to note that the network only
connects beads inside a same protein chain and does not prevent the relative motions between
protomers. The final composition of each system comprised around 20000 atoms, in a simulation
box with dimensions of approximately 15*15*10 nm³. For each system, four independent replicas
were simulated. In each replica, after 10000 energy minimization steps, a 1 ns simulation in the
NVT ensemble was performed, with a time step of 2 fs and position restraints on the protein
backbone beads (force constant of 1000 kJ*mol-1*nm-2) and on the lipid heads (force constant of
200 kJ *mol-1*nm-2). Next, in three consecutive equilibration steps in the NPT ensemble totaling
20 ns, position restraints were maintained but the time step was increased to 4, 10 and finally 20
fs, which was kept in all subsequent simulations. Another 20 ns of equilibration in the NPT
ensemble were performed in 4 more steps, with progressive reduction of the position restraints,
until their complete removal in the last step. Next, the production trajectory, also in the NPT
ensemble, was 10 ms long (considering the 4 replicas, the total production dynamics time was 40
µs per system). The temperature was maintained at 310 K by the v-rescale thermostat100 and the
pressure was maintained at 1 bar by the Berendsen algorithm101. Electrostatic interactions were
treated by reaction-field method, with a cut-off of 1.1 nm and a dielectric constant of 15, and van
der Waals interactions were cut-off at 1.1 nm. The centers of mass of the receptors and the distances
between them were determined with the open-source, community-developed PLUMED library102,
version 2.8. The torsional angle describing the relative orientation between mGlu2 and 5-HT
2A
was determined according to a previously described strategy69 and calculated with PLUMED.

Curve fitting and data analysis
Data in all figures were shown as mean ± SEM from three independent experiments at least and
performed in triplicates. Curves are fitted in Y=Bottom + (Top-Bottom)/(1+10^(LogEC50-X))
using nonlinear regression in GraphPad Prism 8 software (GraphPad Software, San Diego, CA,
USA). Statistical differences were determined using one-way ANOVA with Dunnett’s multiple
comparisons test or two-way ANOVA with Tukey’s multiple comparisons test in GraphPad
Prism8, considering to be significantly different when P-value < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a very elegant manuscript with a massive amount of work testing the residues of the 5-HT2A and mGlu2 receptors that mediate their physical interaction as a GPCR heterocomplex. The techniques involve TR-FRET, advanced computer modeling, cysteine crosslinking, and functional assays using both nanobody-based FRET and other traditional functional assays such as IP1. The authors propose an heteromeric interface between 5-HT2A and mGlu2 mediated via an asymmetric TM4-TM5 interaction, as well as a new allosteric interaction via which 5-HT2A modulates the G protein coupling preferences of the mGlu2 among Go vs Gi.\nComments:\nThe authors use non-cell permeant fluorescent substrates to test FRET (or molecular proximity between 5-HT2A and mGlu2). However, other groups have shown that 5-HT2A has a high level of basal endocytosis and intracellular localization (PMID: 18703670, PMID: 33082287, PMID:36795823). Does the use of non-cell permeant FRET substrates limit the population of 5-HT2A-mGlu2 complexes that is measured?\nCan the authors provide a better explanation of the rationale behind the position of Halo-tag and Snap-tag? If the Halo-tag is located at the N-terminal of mGlu2, is the distance considering the large Venus flytrap extracellular domain of the class C GPCRs short enough to allow FRET with the Spap-tag located also at the N-terminal of the 5-HT2A? The authors mention this at the beginning of the results sections, but the effect of distance between the two N-terminals of mGlu2 and 5-HT2A is not clearly described.\nWhy the ratios of ExpH/ExpS in Fig 1 have a shorter range (less that 2) for the SmGlu2-H5-HT2A experiments, as compared to SmGlu2-HmGlu2 and SmGlu2-HM2R (above 3)?\nStatistical analysis (comparing FRETmax and FRET50) are not assessed in the FRET saturation assays.\nAuthors show FRET signal using the “wild-type” 5-HT2A and mGlu2 receptors in Fig 1. This replicates what others have shown using assays in HEK293 cells under a “basal” state (i.e., without 5-HT2A or mGlu2 agonists) (PMID: 18297054, PMID: 19590495, PMID: 22300836; PMID: 29858599). However, the authors use a constitutively active mutant (mGlu2-Y6.58C, or mGlu2-Ctrl) to conduct all the very elegant cysteine-crosslinking studies. Have the authors tested cysteine-crosslinking with the “wild-type” mGlu2?\nThis is important because as mentioned (and demonstrated) by authors, the structural arrangement of the mGlu2 homodimer is changed upon agonist activation. Previous work by others indicates that TM4 (and in particular three alanine residues located at the intracellular half of the TM4 of the mGlu2: Ala4.40, Ala4.44 and Ala4.48) are necessary for the mGlu2 to form a GPCR complex with 5-HT2A (PMID: 23129762; PMID: 18297054; PMID: 37989895). This has been validated using photoactivatable unnatural amino acids (PMID: 32726588). How can the authors integrate their findings with a constitutively active mGlu2 mutant and previous findings using the wild-type mGluR2?\n5-HT2A also forms homocomplexes (as shown by authors in Figure S4 and by others in PMID: 19279328). Would it be possible to model an alternative arrangement of the GPCR protomers in Fig 4D, showing proximity between the two 5-HT2A protomers?\nAs mentioned, the amount of work for the cysteine crosslinking assays is massive. However, it is not clear (in figure legends or methods) what signal is presented in the representative cross-linking studies. Did the authors use an immunoblot? If so, what antibody did the authors included? Was it targeting 5-HT2A or mGlu2? Again, this is not mentioned in methods, results or figure legends.\nThere is an always experimental variation in among all the SDS-PAGE gel electrophoresis experiments. However, authors present images coming from totally independent gels. This is not usually presented this way (PMID: 12496294, PMID: 16301531). How did the authors control for this inter-experimental variation?\nFig 5a - the cartoon on the left says “inactive” but has a LY379 bound to the Venus flytrap\nFig 5d – Is the buffer – mGlu2+5-HT2A-F173P condition different to the buffer – mGlu2 condition? This comparison is not shown in the figure panel.\nFor crosslinking assays in the presence of drugs, cells were incubated for 30 min, which may induce endocytosis. Was crosslinking observed at the cell surface or intracellularly?\nFig 3F – why the Y-axis goes from 0 to 30 if data are presented as %?\nThe authors elegantly show that 5-HT2A is able to recruit Gq signaling in cells expressing 5-HT2A alone (Fig S9c). Is has also been shown that LY379268-induced activation of mGlu2 trans-activates 5-HT2A and induces Gq dependent signaling (PMID: 29858599; PMID: 26758213). Does the 5-HT2A-F173P mutant prevent this trans-activation (i.e., effect of LY379268 on Gq signaling in cells co-expressing mGlu2 and 5-HT2A as compared to cells co-expressing mGlu2 and 5-HT2A-F173P)\nThe 3D model was carried out before the cryo-EM/X-ray structures of mGlu2 and 5-HT2A were available. How different is the 3D model using these new cryo-EM/X-ray structures?", "reviewer_2": "The manuscript entitled “Allosteric activation of the glutamate receptor mGlu2 by the serotonin receptor 5-HT2A” (NCOMMS-25-07111) seeks to establish oligomerization of the mGlu2 homodimer with a class A GPCR, the serotonin receptor 2A (5-HT2A) as a mechanism for positive allosteric modulation of mGlu2. Initially, Gai et al. use FRET saturation experiments with N-terminally SNAP- and Halo-tagged receptors to prove the interaction of the aforementioned receptors within the plasma membrane of living cells. Again using resonance energy transfer experiments, they show that the dynamic equilibrium between active and inactive mGlu2 is shifted towards the active state in the presence of 5-HT2A, fostering the binding of an active state-specific nanobody. These effects were independent of 5-HT2A activation since incubation with 5-HT2A agonists or antagonists did not significantly alter the observed responses. In contrast, inhibition of mGlu2 with an antagonist reduces the interaction. Furthermore, the authors perform coarse-grained molecular dynamics simulations with a homology model of the putative mGlu2/5-HT2A oligomer, leading to the identification of TMs4 and 5 being likely responsible for the protein-protein interaction. These computational studies inform cysteine-crosslinking experiments with engineered receptor mutants that the authors performed to identify individual residues of the involved receptors that are in close proximity to each other.\nAlthough understanding how cells are able to integrate signals from multiple receptors is relevant, especially for complex structures such as the CNS, I cannot recommend the presented manuscript for publication in Nature Communications for the reasons detailed below:\nContext and scientific rationale\n• There is already a wealth of evidence for the direct interactions between mGlu2 and 5-HT2A in the literature. The manuscript only provides limited advancement to the field. This existing prior knowledge is not adequately reflected (in part not even mentioned):\nMutational studies and domain exchange analyses with mGlu3 already established TMs4 of both mGlu2 and 5-HT2A as most likely interacting domains, even specifying individual residues. The authors also do not comment on prior evidence on the mechanism for allosteric control of 5-HT2A signaling by mGlu2 (e.g. Moreno et al. J Biol Chem 287, 44301-44319, 2012 and Moreno et al. Sci. Signal. 9, ra5, 2016).\n• What is the author’s rationale for using a labeling strategy (SNAP/Halo-tag) that only identifies receptors present on the cell surface, although it has been shown that the majority of 5-HT2A resides in intracellular compartments? It seems that the interaction of 5-HT2A and mGlu2 is a means for controlling mGlu2 surface expression (Toneatti et al. Sci. Signal. 13, eaaw3122, 2020).\n• How do their results compare to previous evidence on hetero-oligomerization of these receptors acquired by BRET saturation and other techniques (e.g. Gonzales-Maeso et al., Nature, 2008, 93–97)?\n• Likewise, it has already been shown that phosphorylation of a specific mGlu2 serine residue by 5-HT2A promotes Gi/o signaling (Murat et al. Mol Psychiatry 24, 1610–1626, 2019).\n• On various occasions, the way scientific background is reported or data are interpreted makes it very complicated to understand the intention of the authors, especially for non-specialized readers. For example, in the introduction it is often unclear which specific homodimers they refer to or whether the authors discuss insights on class A or class C GPCRs, which differ dramatically in their overall architecture and function. Frequently, the authors write about “the two receptors” sometimes meaning to the two protomers of mGlu2 and on other occasions referring to the mGlu2-5-HT2A pair.\nDescription of the methodology\n• The material and methods section lacks critical information that would be needed to reproduce the findings from the study. For example, neither cell densities, nor DNA amounts or at least relative ratios of the individual constructs used for transfections are indicated. How much protein was loaded on the individual blots? How was the protein concentration established? In general, all blots are cropped and missing loading controls.\n• In nearly all Figures, normalized data is presented but there is no information on how normalization was performed.\n• Although coarse-grained MD simulations are beyond the specific expertise of this reviewer, it must be stated that the description of the experimental procedure lacks critical details that are needed to fully understand or yet reproduce the authors’ experiments. For instance, there are no statements on how the authors dealt with the G proteins present in the cryo-EM structures used as templates, and neither did they comment on the large venus-fly trap domains present on mGlu2. Assuming that the G proteins were not part of the simulations, how did the authors ensure that their simulated “active states” were true active states without setting any constraints on the G protein-binding interfaces?\nTechnical aspects\n• The authors use homology models for their MD simulations and saying that their study was initiated before high-resolution structures of mGlu2 and 5-HT2A were available. They state: “More recently, we have further validated this model through a new complex model built from the cryo-EM structures […]”. Is there any data supporting this claim?\n• The authors use M2 as a negative control in their FRET saturation assays. Is there any evidence suggesting that this receptor does not interact with mGlu2? In fact, the FRET curve with M2 shows a nice saturation profile. The FRET signal is lower than with 5-HT2A but this could be an effect of different distances between the SNAP and Halo-tags, given that 5-HT2A has a much longer N-terminus (~68 AS) than M2 (~18AS). A more appropriate experiment to study the specificity of the interaction could be FRET competition assays with non-labeled 5-HT2A.\n• Line 271: “Interestingly, the mGlu2 receptor activated by the 5-HT2A receptor can no longer induce Go protein rearrangement but can still induce Gi protein rearrangement.” - This conclusion is not valid. Once activated by an agonist, mGlu2 induces activation of Go, also in the presence of 5-HT2A. In contrast, there is a much smaller add-on effect of mGlu2 agonist on the signaling of Gi3 (Fig 6c).\n• In Figure 1b, the authors draw a 2:1 stoichiometry of mGlu2 and 5-HT2A – could it also be 2:2 stoichiometry? Why is it drawn in 2:2 in Fig. 3d? In addition, there is a mistake in labeling: the Halo-tag of the high-FRET state (middle) does not glow, while in the low FRET state it glows (bottom).", "reviewer_3": "Gai and colleagues study mGlu2-5HT2a heterodimers using TR-FRET, oxidative crosslinking, modeling and BRET-based G protein activation assays. The main conclusion of the study is that 5HT2a acts as a positive allosteric modulator, capable of binding to and thus stabilizing the active conformation of the 7TM domains of mGlu2 dimers. Interestingly, the authors conclude that 5HT2a promotes mGlu2 activation of Gi but not Go heterotrimers, the latter being the predominant G protein expressed in neurons. While most of the data presented in the manuscript are consistent with this conclusion, the authors present little evidence that directly supports the model or rules out alternatives. There are also internal inconsistencies and conflicts with prior work that the authors do not address, as well as some technical shortcomings that undermine confidence in the work. In its present form the manuscript contains some interesting data but isn’t a complete and compelling report.\nThe strongest evidence supporting the authors’ model is shown in Figure 5b, which suggests that 5HT2a can drive mGlu2 towards the active state, apparently almost to the same extent as LY379268. The assay used here requires overnight incubation with ligands and nanobodies (Nb) at room temperature. As expression data are normalized there is no way to know if surface expression of mGlu2 changes as 5HT2a is increased, which could produce an increase in TR-FRET independent from receptor activation. Moreover, expression is said to be assessed using Tb emission after labeling of SNAP or Halo, therefore expression data presumably do not come from the same samples as the ones binding DN1-Tb. It is also unclear to what extent the assay is affected by the DN10 Nb itself, since this nanobody binds to the active state of the extracellular domain. This experiment is not controlled using another class A receptor that does not interact with mGlu2 and is described “triplicates from a typical experiment”, i.e. the results of one experiment. While intriguing, this result needs better controls and support from an orthogonal assay. One obvious alternative would be TR-FRET between SNAP and Halo tagged mGlu2 protomers, which the authors (and others) show is acutely sensitive to agonists and antagonists. This assay would allow the authors to combine expression of 5HT2a and real-time pharmacology using single samples.\nSignaling studies using Gqi9 as a surrogate for Gi/o heterotrimers do not strongly support robust positive allosteric modulation of mGlu2 by 5HT2a. The enhancement due to 5HT2a F173P shown in Figure 5f is rather modest with n=3, and Figure S9a shows no change in the LY379268 EC50. The mGlu2/5HT2a expression ratio is not given here, mGlu2 surface expression is not quantified, and there is no negative control class A receptor.\nBRET studies to assess heterotrimer activation show a large negative shift in the baseline when 5HT2a is expressed for Gi3 but not Go. The authors optimistically interpret this (in Figure 6b) as an increase in mGlu2-mediated activation of Gi3, but do not consider how this baseline shift can be much larger than the effect of a full agonist (Figure S10a). Since these assays are sensitive to overall expression of donors and acceptors it is much more likely that the baseline shift is an artifact of adding another overexpressed protein (5HT2a). Moreover, the agonist-induced BRET decrease for Gi3 is smaller in the presence of 5HT2a (Figure S10 and 6c) making it unclear if 5HT2a increases or decreases (or does not change) the ability of mGlu2 to activate Gi3. Clearly 5HT2a does not occlude Gi3 activation in this assay.\nIt is concerning that none of these BRET changes occur for GoB. The authors simply accept this at face value as evidence for 5HT2a-induced bias between closely-related G protein subtypes. Overall the BRET studies do not clearly support the authors’ model and indeed raise some doubts.\nA large portion of the manuscript is devoted to “saturation” TR-FRET studies and oxidative crosslinking studies meant to provide evidence for the existence of mGlu2-5HT2a heterodimers and the relevance of a particular interprotomer interface. These studies are only tangentially related to the model the authors are proposing.\nThe crosslinking studies provide data that are consistent with the structural model, and the amount of work represented by these studies is appreciated. However, the results ultimately show that a TM4/5 interface for mGlu2 becomes accessible for crosslinking when this receptor is active, which could have been inferred from the already-available structures. The information gained here isn’t leveraged for functional studies. Again, the crosslinking studies aren’t controlled by any other class A GPCR.\nGiven the long history of this heterodimer (first described in 2008) the saturation TR-FRET studies cover old ground using methods with well-known technical limitations. A “saturating” curve is uninterpretable if acceptor density is normalized using donor density, and changes in response to agonist (as in Figure 3a) could reflect conformation as much as association. Agonist-induced changes are not controlled by any other TM protein. Here again there are unexplained inconsistencies. In the authors’ model some 5HT2a interacts with unliganded mGlu2 because of constitutive activity or ambient glutamate. Why then does agonist apparently enhance “association” with 5HT2a (Fig. 3a), but antagonist does not do the opposite (S2b).\nThe functional studies rely on a defective 5HT2a mutant F173P, which is described as a mutation in the intracellular part of TM3 that blocks G protein coupling. The rat 5HT2a sequence has no F residues in the intracellular part of TM3, and residue 173 is the R in the middle of the DRY motif. No citation is provided for whatever this mutant is, and the methods section on plasmid construction is also unhelpful. When used it is not clear than this mutant receptor was expressed at the cell surface, and validation for loss of function is minimal. It is not clear that Gqi9 is present in Figure 5e, which is necessary to interpret Figure 5F.\nThroughout the manuscript there are instances of questionable experimental rigor. For example, in Figure 1 we are told “Data are means ± SEM of triplicates from a typical experiment”, which suggests three technical and one biological replicate. Similar descriptions appear in Figures 3a, 5b, S2, etc.. The legend for Figure 6b says 3 independent experiments but there are 10-12 data points on the bars, suggesting the authors are plotting technical replicates. Since an ANOVA is performed on these data it’s not clear what they are including. In Fig. 6c there are 6 points on the graph for “a typical experiment performed at least 3 times”.\nThis heteromeric complex has been studied for almost 20 years but the authors provide scant discussion to place their findings in context of what is already known. For example, how can these observations be reconciled with a report that mGlu2-dependent activation of Gi/o is unchanged in 5HT2a -/- mice (PMID: 26758213).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Rebuttal point-by-point\nReviewer #1:\nThis is a very elegant manuscript with a massive amount of work testing the residues of the 5-\nHT2A and mGlu2 receptors that mediate their physical interaction as a GPCR heterocomplex. The\ntechniques involve TR-FRET, advanced computer modeling, cysteine crosslinking, and functional\nassays using both nanobody-based FRET and other traditional functional assays such as IP1. The\nauthors propose an heteromeric interface between 5-HT2A and mGlu2 mediated via an\nasymmetric TM4-TM5 interaction, as well as a new allosteric interaction via which 5-HT2A\nmodulates the G protein coupling preferences of the mGlu2 among Go vs Gi.\nWe wish to thank the referee for his/her very positive view of our study. As detailed below, we\ndid our best to clarify all the critics raised on our work.\nComments:\nThe authors use non-cell permeant fluorescent substrates to test FRET (or molecular proximity\nbetween 5-HT2A and mGlu2). However, other groups have shown that 5-HT2A has a high level\nof basal endocytosis and intracellular localization (PMID: 18703670, PMID: 33082287,\nPMID:36795823(Lopez-Gimenez et al., 2008; Toneatti et al., 2020; Vargas et al., 2023)). Does\nthe use of non-cell permeant FRET substrates limit the population of 5-HT2A-mGlu2 complexes\nthat is measured?\nTo clarify this point, we did examine the localization of either mGlu2 and 5-HT receptors fused\n2A\nto RFP and eGFP, respectively, in the cells used for our study. We compared our conditions with\nthe one described in these three references (Rebuttal-Table 1). In our study, in basal conditions\nthe rat 5-HT -eGFP receptor construct expressed alone is found mainly at the level of the plasma\n2A\nmembrane, but some intracellular 5-HT receptors can be observed (Rebuttal-Figure 1a). This\n2A\nis in contrast to these previous studies where there is a strong intracellular localization of the\nrecombinant human 5-HT receptor in basal conditions (Toneatti et al Sci Signal (2020) PMID\n2A\n33082287 ; Lopez-Gimenez et al Mol Pharm (2008) PMID 18703670 ; Vargas et al Science (2023)\nPMID 36795823). These differences with the previous studies might be due to the difference in\nthe constructs, including the signal peptide used in our study, or of the conditions of transfection\nand expression (Rebuttal-Table 1). But interestingly, when our rat 5-HT -eGFP construct is co-\n2A\ntransfected with the rat mGlu2-RFP construct, 5-HT receptors are no longer observed\n2A\nintracellularly (Rebuttal-Figure 1c), suggesting mGlu2 may help most of the 5-HT receptors to\n2A\nreach the cell surface.\nOf note, upon 5-HT agonist activation, a larger amount of intracellular 5-HT -eGFP receptors\n2A\ncan be detected when expressed alone (Rebuttal-Figure 1a) or co-transfected with mGluR2\n(Rebuttal-Figure 1c), with 5-HT receptors co-localized with mGlu2 in some of the intracellular\n2A\ncompartments. This suggests that some of the 5-HT receptors can co-internalize with mGlu2,\n2A\nconsistent with a fraction of these receptors being interacting.\nIn contrast, our rat mGlu2-RFP construct expressed alone is localized mostly at the plasma\nmembrane (Rebuttal-Figure 1b), and we did not observe detectable mGlu2 internalization upon\nagonist activation, in agreement with previous studies (Abreu et al., Cell Rep 2021 – PMID\n33910009; Cimadevila et al., Cell Rep 2025 – PMID 40748754).\nIn our FRET study, although we used different constructs (Halo-tagged rat 5-HT and SNAP-\n2A\ntagged rat mGluR2 constructs with unmodified C-terminal regions), we believe that under our\n1\nexperimental condition, most of the receptors are similarly and mostly at the cell surface, and\ntherefore, are prone to be labeled with the non-cell permeant SNAP and Halo substrates.\nHowever, such conclusions would need further experiments that are, we think, out of the scope of\nthe present study as they are not needed for our main conclusion.\nmGlu2 alone 5-HT alone mGlu2 + 5-HT Reference Remarks\n2A 2A\nLow High High for both Toneatti - Human cMyc-5-HT -\n2A\nmGlu2 and 5- (2020) mCherry or YFP\nHT 2A PMID - Flip-in HEK293\n33082287 inducible stable\ntransfection\n- Induced for 24-48 h\nN.D. High N.D. Lopez- - Human cMyc-5-HT -\n2A\nGimenez eCFP\n(2008) - Flip-in HEK293\nPMID inducible stable\n18703670 transfection\n- Induced for 24 h\nN.D. High N.D. Vargas - cMyc-5-HT -eCFP\n2A\n(2023) - HEK293T - 24h after\nPMID transient transfection\n36795823\nLow Low Low for both our - Halo or SNAP-tagged\npresent receptors\nstudy - Transient transfection for\n18 h in HEK293 cells\nRebuttal Table 1. Comparison of the levels of mGlu2 and 5-HT receptors intracellularly\n2A\nretained in the indicated basal conditions. N.D., not determined.\n2A\nin basal or agonist induced conditions. Fluorescence detection of 5-HT -eGFP and mGlu2-RFP in transfected\n2A\nHEK293 cells after incubation with control buffer, LY379268 (20 M) mGlu2 agonist or serotonin (5-HT, 10\nM) 5-HT agonist. Before treating cells with drugs, they were incubated for 4 h in Glutamax medium without\n2A\nserum and then starved with PBS for 0.5 h. Images were taken after a 30 min incubation with or without the\nindicated agonist.\nCan the authors provide a better explanation of the rationale behind the position of Halo-tag and\nSnap-tag? If the Halo-tag is located at the N-terminal of mGlu2, is the distance considering the\nlarge Venus flytrap extracellular domain of the class C GPCRs short enough to allow FRET with\nthe Snap-tag located also at the N-terminal of the 5-HT2A? The authors mention this at the\nbeginning of the results sections, but the effect of distance between the two N-terminals of mGlu2\nand 5-HT2A is not clearly described.\nIt is difficult to give a precise distance between the fluorophores attached to the Snap and Halo\ntags fused to the N-terminus of each receptor, as both are linked via a flexible linker (see Supp\nFig. 4 in Olofsson et al Nat Comm 2014 – PMID 25323157). It is true that the distance between\nthe N-terminus of mGlu2 (large extracellular domain of 563 amino-acids) and the surface of the\nplasma membrane is close to 9 nm, a distance quite large not expected to give a significant FRET\nwith the short N-terminus of 5-HT (68 amino-acids). This is why we attached the largest tag\n2A\n(Halo-tag; 33 kDa) to the N-terminus of 5-HT and the Snap-tag (20 kDa) to the N-terminus of\n2A\nmGluR2, to bring up the fluorophores attached to 5-HT , reaching a distance compatible with\n2A\nFRET between fluorophores.\nAs suggested by the reviewer, we compared the FRET signal between the two receptors when the\nHalo-tag fused to the N-terminus of mGlu2 was co-transfected with a 5-HT receptor fused with\n2A\nSnap tag at its N-terminus. A smaller FRET signal was measured (Rebuttal Figure 2) compared\nto the use of Snap-tagged mGluR2 co-transfected with Halo-tagged 5-HT (Figure 1b). In\n2A\naddition, we show that the FRET efficacy is dependent with the pair of fluorophores used. As\nexpected, using a pair with a smaller Ro (50 Å for Lumi4Tb and Green) give a smaller signal than\na pair with a larger Ro (58 Å for Lumi4Tb and Red; Scholler et al., Nat Chem Biol 2017 – PMID\n28135236). Finally, the large FRET signal used with the fluorophore pair Lumi4Tb and Green\n3\nshowed that the distance between the Snap-tag attached to mGlu2 and Halo-tag linked to 5-HT\n2A\nis within the 50 Å range (Figure 1b).\nRebuttal Figure 2: Intensity of the FRET signal at the cell surface between HmGlu2 and either S5-HT or SM2\n2A\nreceptors in cotransfected HEK293 cells, after colabeling with the non-cell permeant fluorophores Halo-Tb as\ndonor and either SNAP-Red or SNAP-Green as acceptors. The expression of HmGlu2 (Exp ) and S5-HT at\nH 2A\ncell surface was measured by the emission signal of Halo-Tb or Snap-Tb, after individual labeling of separate\ncell batches. The expression ratio of S5-HT / HmGlu2 and SM2 / HmGlu2 are is 1.24 and 1.66, respectively.\n2A\nData are mean ± SEM from a typical experiment performed three times (n=3).\nThese data are presented in the new Supplementary Figure 1 of the revised manuscript.\nWhy the ratios of ExpH/ExpS in Fig 1 have a shorter range (less that 2) for the SmGlu2-H5-HT2A\nexperiments, as compared to SmGlu2-HmGlu2 and SmGlu2-HM2R (above 3)?\nThis is due to a lower expression of the Halo-5-HT receptor, compared to Halo-mGlu2 or\n2A\nHalo-M2 receptors. This is now better stated in our revised manuscript.\n5-HT and M2) in three independent experiments. (a, d, g) FRET saturation analysis between SmGlu2 and\n2A\nHmGlu2, H5-HT or HM2 coexpressed at the cell surface of HEK293 cells as indicated. (b, e, h) Expression\n2A\nlevel of SmGlu2 at cell surface when coexpressed with HmGlu2, H5-HT or HM2. (c, f, i) Expression level of\n2A\nHmGlu2, H5-HT or HM2 at cell surface when co-expressed with SmGlu2. A fixed amount of SmGlu2 (20 ng)\n2A\nwas cotransfected with increased amounts (from 0 to 80 ng) of HmGlu2, H5-HT or HM2, as indicated.\n2A\nExpression levels were normalized to SmGlu2 expressed alone in the positive control (SmGlu2 + HmGlu2).\nExperiments 1, 2 and 3 are shown in (a-c), (d-f) and (g-i), respectively.\nThese data are presented in the new Supplementary Figure 2 of the revised manuscript.\nStatistical analysis (comparing FRETmax and FRET50) are not assessed in the FRET saturation\nassays.\nWe have plotted these values from the three independent TR-FRET saturation experiments above.\nThese data were analyzed in one-site specific binding. We can provide the values of Bmax and K\nD\nwhich are similar to FRET Max or FRET respectively (See Rebuttal Table 2). We found a\n50\nsimilar K values for the different receptors, but the estimated Bmax values are statistically\nD\ndifferent.\nRebuttal Table 2: Summary of the Best fit values of Bmax and K for three independent TR-\nD\nFRET saturation experiments.\nN=1 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.06 2.00 0.90\nK 0.48 0.73 0.51\nD\nN=2 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.17 1.22 0.49\nK 0.75 0.22 0.43\nD\nN=3 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.52 2.53 0.98\nK 0.57 0.88 0.76\nD\nAuthors show FRET signal using the “wild-type” 5-HT2A and mGlu2 receptors in Fig 1. This\nreplicates what others have shown using assays in HEK293 cells under a “basal” state (i.e., without\n5-HT2A or mGlu2 agonists) (PMID: 18297054, PMID: 19590495, PMID: 22300836; PMID:\n29858599(Gonzalez-Maeso et al., 2008; Rives et al., 2009; Delille et al., 2012; Murat et al., 2019)).\nHowever, the authors use a constitutively active mutant (mGlu2-Y6.58C, or mGlu2-Ctrl) to\nconduct all the very elegant cysteine-crosslinking studies. Have the authors tested cysteine-\ncrosslinking with the “wild-type” mGlu2?\nThis is important because as mentioned (and demonstrated) by authors, the structural arrangement\nof the mGlu2 homodimer is changed upon agonist activation. Previous work by others indicates\nthat TM4 (and in particular three alanine residues located at the intracellular half of the TM4 of\nthe mGlu2: Ala4.40, Ala4.44 and Ala4.48) are necessary for the mGlu2 to form a GPCR complex\nwith 5-HT2A (PMID: 23129762; PMID: 18297054; PMID: 37989895(Gonzalez-Maeso et al.,\n2008; Moreno et al., 2012; Gao et al., 2024)). This has been validated using photoactivatable\nunnatural amino acids (PMID: 32726588(Shah et al., 2020)). How can the authors integrate their\nfindings with a constitutively active mGlu2 mutant and previous findings using the wild-type\nmGluR2?\nOur data highlight the fact that 5-HT receptor preferentially interact with the active forms of\n2A\nmGlu2, a conformation that exposes the TM4-5 interface (PDB 7MTS) where the 5-HT\n2A\ninteracts. Accordingly, WT mGlu2 and 5-HT can indeed interact, as illustrated in Figure 1b.\n2A\nWhen using Cys-crosslinking experiments, we did test the WT mGlu2 and observe a weak cross-\nlinking (this was indeed shown in Supplementary Fig 3c,d,f in our original paper; see below as\nRebuttal Figure 4). We interpret this as a difficulty of the two proteins to orient correctly due to\nthe structural dynamics of the mGlu2 receptor likely oscillating from the inactive to the active\nstate, limiting the possibility of a disulfide bound formation. This is the reason why we used a\nmGlu2 receptor mutant stabilized in its active state, making the TM4-5 always available for\ninteraction with the 5-HT receptor. Under such condition, a higher crosslinking could be\n2A\nobserved (New Figure 3g-h). This is perfectly consistent with our model.\nRebuttal Figure 4. (a) Cross-linking of the indicated constructs of S5-HT mutant and HmGlu2 wild-type or\n2A\nmutant after labeling with non-cell permeable fluorescent Snap-Alexa Fluor 782 substrates. The mutant\nconstructs contain a single cysteine at positions mGlu25.42 and 5-HT 5.41. The results were obtained after pre-\n2A\nincubation with mGlu2 receptor agonist LY379268 and CuP. (b) Change of the percentage of cross-linked\nmGlu2-5-HT hetero-oligomers at the cell surface in the presence or absence of LY379268 (10 M). The\n2A\namount of S5-HT subunits labeled at the cell surface by the Snap-Surface Alexa 782 substrate was quantified\n2A\nby emission at 800 nm. Data are mean ± SEM from at least three independent experiments. One-way ANOVA\nwith Dunnett’s multiple comparisons test with ***P < 0.001 and ns, not significant. (c) SDS-PAGE analysis of\nthe mGlu2-5-HT hetero-oligomer. Detection of the indicated co-expressed S5-HT and HmGlu2 wild-type or\n2A 2A\nmutants after labeling with non-cell permeable fluorescent substrates. Snap-tag and Halo-tag were labeled with\nSnap-Alexa Fluor 782 and Halo-Alexa Fluor 660, respectively. The cross-linked mGlu2-5-HT complex can\n2A\nbe specifically detected after introducing cysteine mutations in 5.41 of 5-HT and 5.42 of mGlu2.\n2A\nPanels (a) and (b) are presented in the New Figure 3 of the revised manuscript, and panel (c) is\npresented in the Supplementary Figure 10 of the revised manuscript.\n6\n5-HT2A also forms homocomplexes (as shown by authors in Figure S4 and by others in PMID:\n19279328(Brea et al., 2009b)). Would it be possible to model an alternative arrangement of the\nGPCR protomers in Fig 4D, showing proximity between the two 5-HT2A protomers?\nIn our cross-linking experiments, two conformational arrangements for the 5-HT homodimer\n2A\nwere found involving either TM4-5 or TM1 interfaces (Supplementary Figure 14). These results\nagree with the study by Brea et al. (Brea et al. (2009) Mol Pharm – PMID 19279328), who\npresented data supporting the existence of 5-HT receptor homodimers in live cells by\n2A\ncoimmunoprecipitation studies and FRET experiments. Interestingly, they proposed a\nmathematical model consisting of a three-state preformed homodimeric receptor, including an\ninactive state, R2, and two active states, R2* (inositol phosphate signaling pathway) and R2**\n(arachidonic acid signaling pathway), where the active receptor states were selectively\ndistinguished by typical and atypical antipsychotics. It is worth noting that, as the authors\nacknowledged, their data were also compatible with a dynamic equilibrium between monomeric\nand dimeric 5-HT receptors showing different functionality (i.e., monomeric receptors signaling\n2A\nvia the inositol pathway and dimeric receptors signaling via the arachidonic acid pathway, the\nlatter displaying ligand-dependent cooperativity).\nWe appreciate the suggestion of the reviewer, and we have modeled a receptor complex consisting\nof the mGlu2 obligate homodimer and a 5-HT homodimer to fit Figure 4d of our manuscript.\n2A\nOf the two conformational arrangements provided by our crosslinking experiments for the 5-HT\n2A\nhomodimer, only the one featuring the TM1-TM1 interface is consistent with the proposed mGlu2-\n5-HT heteromeric model (Rebuttal Figure 5). As such, a 5-HT dimer composed of subunits\n2A 2A\ninteracting via TM4-5, are unlikely able to interact with mGlu2. It is however our view that the 5-\nHT receptor dimers are likely dynamics, oscillating between a monomeric and a dimeric state,\n2A\nthen making possible the interaction of the 5-HT receptor with the active form of mGlu2\n2A\nhomodimer.\nRebuttal Figure 5. mGlu2-5-HT heteromeric receptor model including a 5-HT homodimer. The TM1-\n2A 2A\nTM1 interface for the 5-HT homodimer is compatible with the heteromerization interface in mGlu2-5-HT\n2A 2A\ncomplex based on the cross-linking results.\nThese data are presented in the new Supplementary Figure 14 of the revised manuscript.\nThese studies are only tangentially related to the model the authors are proposing.\nWe agree with the referee, but because a few laboratories questioned the veracity of the previously\nreported data coming from a unique laboratory, we still think it was important to confirm this\ninteraction using different approaches. Validating these proposals was indeed essential to reach\nour aim that 5-HT receptors could potentially activate mGlu2 by interacting with an interface\n2A\nonly accessible in the active form of the receptor. Of importance, our data are the first to\ndemonstrate the conformational dependence of the mGlu2-5-HT receptor interaction. In our\n2A\nview, this is a very important and novel finding.\nThe crosslinking studies provide data that are consistent with the structural model, and the amount\nof work represented by these studies is appreciated. However, the results ultimately show that a\nTM4/5 interface for mGlu2 becomes accessible for crosslinking when this receptor is active, which\ncould have been inferred from the already-available structures. The information gained here isn’t\nleveraged for functional studies. Again, the crosslinking studies aren’t controlled by any other\nclass A GPCR.\nThe experiments were designed and done before the mGlu2 structures were available, and based\non our hypothesis for mGlu2 activation (Xue et al., NCB 2015 – PMID 25503927), a model that\nwas not accepted by many groups as the intersubunit movement was judged too big. Since then,\nour proposal has been validated by the cryoEM structures of mGlu2 in various states. Although\nthe mGlu inactive state structures show 7TM subunit often facing each other via TM4-5, this is\nnot always the case (see mGlu3 inactive states in Fang et al., Cell Res 2022 – PMID 35236939),\nand this very static view is unlikely real, as the 7TMs are likely to move one compared to the other,\ngiving some possible flexibility for a class A GPCR to interact with the TM4-5 interface. Of note,\nthe very different 7TM relative positions in the various mGlu inactive state structures (see\nstructures of mGlu1, 2, 3 and 5 in the inactive state; also reviewed by Liu et al (2023) Am J Physiol\nCell Physiol – PMID 37184233) support such a constant dynamic of the two 7TM domains in the\ninactive conformation.\nThe reviewer asked about the crosslinking studies by any other class A GPCR. We believe that the\nbest negative control for the relevant crosslinking are the absence of cross-linked observed\nbetween mGlu2 TM4-5 and TM1 and TM6 of 5-HT receptor (because TM1 is also involved in\n2A\nhomodimer interface) (see Supplementary Figure 11).\nGiven the long history of this heterodimer (first described in 2008) the saturation TR-FRET studies\n32\ncover old ground using methods with well-known technical limitations. A “saturating” curve is\nuninterpretable if acceptor density is normalized using donor density, and changes in response to\nagonist (as in Figure 3a) could reflect conformation as much as association. Agonist-induced\nchanges are not controlled by any other TM protein. Here again there are unexplained\ninconsistencies. In the authors’ model some 5HT2a interacts with unliganded mGlu2 because of\nconstitutive activity or ambient glutamate. Why then does agonist apparently enhance\n“association” with 5HT2a (Fig. 3a), but antagonist does not do the opposite (S2b).\nRegarding the original Fig 3a, the referee is correct, this can come either from an increased mGlu2-\n5-HT complexes, but this can also come from a slight change in distance between the\n2A\nfluorophores due to the conformational change in the VFTs. This is now clearly mentioned in the\nResults section (page 8), and used as an argument to directly test the agonist dependency of the\ninteraction using cross-linking experiments.\nIn the new Figure 3, we now include the essential data that were originally shown in Supp Fig\n3c,d, showing a very low cross-linking in the absence of mGlu2 agonist, while a large amount of\ncrosslinked receptors are observed in the presence of the mGlu2 agonist. It is likely that the\ncrosslinking occurs when the interaction is stable enough to allow the two thiols groups to find the\ntime to be in close proximity and in the correct orientation for the disulfide bond to form. However,\nshorter transient interaction between mGlu2 and 5-HT could occur thanks to the constant\n2A\ndynamics of the mGlu2 7TM dimer that likely exists when both VFTs are stabilized open with an\nantagonist. Indeed, although the TM domains are well constraint and always found to interact via\nTM6 in all active state class C GPCRs, the relative position of the two 7TMs are very variable in\nthe apo or antagonist bound states (see structures of mGlu1, 2, 3 and 5 in the inactive state; also\nreviewed by Liu et al (2023) Am J Physiol Cell Physiol – PMID 37184233). It is therefore not\ntotally surprising that the basal FRET observed in the original Supp Fig 2b is not inhibited by\nLY341495. This point is now discussed in the revised manuscript.\nThe functional studies rely on a defective 5HT2a mutant F173P, which is described as a mutation\nin the intracellular part of TM3 that blocks G protein coupling. The rat 5HT2a sequence has no F\nresidues in the intracellular part of TM3, and residue 173 is the R in the middle of the DRY motif.\nNo citation is provided for whatever this mutant is, and the methods section on plasmid\nconstruction is also unhelpful. When used it is not clear than this mutant receptor was expressed\nat the cell surface, and validation for loss of function is minimal. It is not clear that Gqi9 is present\nin Figure 5e, which is necessary to interpret Figure 5F.\nWe are very sorry for this mistake, yes, position 173 in the 5-HT receptor corresponds to the R\n2A\nof the DRY motif. This mistake has been corrected in the entire manuscript, as well as in every\nfigures and Supp figures. This R residue is highly conserved in class A GPCRs (Weis & Kobilka\nAnn Rev Biochem (2018) – PMID 29925258; Rovati et al (2007) Mol Pharm – PMID 17192495).\nAlthough this mutation of R173 into P has not been reported to our knowledge, mutation of this R\nresidue has been studied for many class A GPCRs such as the angiotensin AT1 receptor\n(Suonivuori et al Lefkowitz (2020) Science). How this mutant has been generated is now better\nexplained in the Methods section (Page 20). We also refer to other studies in which mutation of\nthis R residue from the DRY motif suppresses G protein signaling (Rovati et al (2007) Mol Pharm\n– PMID 17192495).\nThis mutation prevents G protein activation by this receptor, as well illustrated in the original Supp\nFig 9c using the G protein biosensors, and in the original Fig 5e, when IP production is measured,\n1\neven in the presence of over-expressed Gqi . As indicated below, this mutant receptor expresses\n9\nvery well at the cell surface (Rebuttal Figure 19), an information now added in the revised paper.\n80000\ne\nc\na\nfr 60000\nu\ns\nlle\nc\nta 40000\nn\no\nis\ns\ne 20000\nr\np\nx\nE\nP\nS m H5- T2 A\n+ H\nGl\nu 2\n+\nH5-\nS m u 2\nGl\nm\nS\nRebuttal Figure 19. Expression level of H5-HT wild-type and R173P mutant at the cell surface in the\n2A\nFigure 5d. The amounts of H5-HT and H5-HT R173P at cell surface were measured by the emission signal of\n2A 2A\nHalo-Tb. H5-HT R173P mutant showed similar expression level to wild-type H5-HT in presence of co-\n2A 2A\ntransfected SmGlu2. Data are mean ± SEM from a typical experiment performed three times (n=3).\nWe now include additional experiments with this mutant, looking at the effect of mGlu2 on the\nactivity of the 5-HT receptor, looking at its capacity to generate a calcium signal (see Rebuttal\n2A\nFigure 6, page 10 of this rebuttal). We show that indeed, when 5-HT receptors are co-expressed\n2A\nwith mGlu2, the mGlu2 agonist LY379268 generates a calcium signal, an effect not observed with\nthe R173P mutant (data are show in our answer to referee #1 in Rebuttal Figure 6, page 10 of\nthis rebuttal). And, the original Fig. 5e-f have been modified to clarify that Gqi has been used in\n9\nthe experiments illustrated in panels e and g in the new Figure 5.\nThroughout the manuscript there are instances of questionable experimental rigor. For example,\nin Figure 1 we are told “Data are means ± SEM of triplicates from a typical experiment”,\nwhich suggests three technical and one biological replicate. Similar descriptions appear in Figures\n3a, 5b, S2, etc.. The legend for Figure 6b says 3 independent experiments but there are 10-12 data\npoints on the bars, suggesting the authors are plotting technical replicates. Since an ANOVA is\nperformed on these data it’s not clear what they are including. In Fig. 6c there are 6 points on the\ngraph for “a typical experiment performed at least 3 times”.\nWe clarified the Fig. 1b, 3a, 5b, Supp Fig 2a by indicating that the data shown are means +/- SEM\nare from three independent experiments (n=3).\nFor Fig. 6b, six independent experiments have been performed for LY37 and buffer conditions,\nwhen mGlu2 is expressed alone or coexpressed with 5-HT mutant. Similarly, for Fig. 6c, six\n2A\nindependent experiments have been performed for LY37 and buffer conditions, when mGlu2 is\nexpressed alone or coexpressed with 5-HT mutant.\n2A\nWe now carefully checked that the number or replicates and number of independent experiments\nare correctly mentioned in every figure legends.\ndiscussion to place their findings in context of what is already known. For example, how can these\nobservations be reconciled with a report that mGlu2-dependent activation of Gi/o is unchanged in\n5HT2a -/- mice (PMID: 26758213(Moreno et al., 2016)).\nWe tried to improve our discussion, respecting the size limit, better referring to the previous\npublished work, done almost exclusively by the group of Javier Gonzales-Maeso. Indeed, we do\nthink it was important to confirm his data using eventually different approaches, as these were\nessential to validate our hypothesis that 5-HT receptor would stabilize the active conformation\n2A\nof the mGlu2 dimer. Our aim was not to go further into the physiological meaning, as these were\nlargely discussed in relation to the antipsychotic effects of mGlu2 and 5-HT receptor ligands by\n2A\nJavier. Regarding the specific point raised by the referee: even though we do believe the 5-HT\n2A\nreceptor can indeed regulate the activity of mGlu2, such a regulation may occur in some specific\nbrain area or neuronal subdomains, such that only a very small fraction of the mGlu2 receptors\ncan be regulated by 5-HT receptors. This is expected as most 5HT receptors are post-synaptic,\n2A 2A\nwhile the mGlu2 receptors are pre-synaptic. In addition, we do believe this effect is transient,\nsuch that even for mGlu2 receptors that can be regulated by 5-HT , only a fraction of these will be\n2A\nassociated with 5-HT at a given time. Taking these points together, one is not expecting that the\n2A\ndeletion of 5-HT receptor will significantly affect the capacity of mGlu2 to regulate Gi/o\n2A\nproteins. This does not exclude an important role of this regulation occurring at specific sites\nimportant for the antipsychotic effects of mGlu2 or 5HT ligands.\n2A\n35"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my previous comments except one: the potential role of residues Ala4.40, Ala4.44, and Ala4.48 in mGluR2 heteromerization with 5-HT2A. This point is important to evaluate the validity and replicability of their findings, as it has been reported by several independent groups (PMID: 23129762; PMID: 37989895).", "reviewer_2": "I am happy that the authors have addressed most of my comments with their revised version of the manuscript “Allosteric activation of the glutamate receptor mGlu2 by the serotonin receptor 5-HT2A”.\nI have one remaining query that relates to my original comment on the missing comparison of the 5-HT2A-mGlu2 homology model with the model built on the available high-resolution structures of the individual receptors:\nWhile the authors now provide an overlay (in top view) of the simulation results from their homology model and the model obtained from experimental data, their conclusions on this comparison do not seem convincing. The new Supplementary Figure 5 shows obvious differences in the positioning of transmembrane helices 4 (and 5) of the 5-HT2AR relative to the mGlu2 protomers, a fact that the authors themselves admit within their experimental section:\n“One difference between the two models is the position of TM4 of the 5-HT2A receptor. In homology model, this helix is located between TM5 of one mGlu2 protomer and TM6 of the other protomer. While in the new model, 5-HT2A TM4 moves towards to the TM5 of mGlu2 protomer, which explains the higher number of frequent contacts between mGlu2 TM5 and 5-HT2A TM4 observed in this case (Supplementary Fig. 7-8).”\nIn their main text and their point-by-point reply, however, they conclude: “Both displayed the same general orientation of the protomers (Supplementary Fig. 5a), with several common contacts at the interfaces (Supplementary Fig. 5b).”/ “It shows that the relative orientation of the protomers is the same in the two models, with a common mGlu2-5-HT2A interface.”\nFirst, an additional view from the side could allow the reader to better assess the major differences in the conformations along helices 4 and 5 of 5-HT2AR. Second, in their point-by-point reply, Supplementary Fig. 5b is said to provide “the full lists of contact frequencies for both models”. However, this Figure only contains a list of amino acids labeled as “frequently observed in CGMD simulations of both heterotrimer models”, with no information on how “contacts” were defined, and what is meant by “frequently”. What were the additional contacts with the model derived from the experimental structures? Furthermore, what is the basis of the order of amino acids in this list? Third, while contact residues for the new model are highlighted in Supplementary Fig. 7e (although hardly visible), there is no data that allows comparison of the contacts between the two models. Unfortunately, the mentioned Supplementary Figure 8 shows something completely different (TR-FRET saturation analysis). The analysis of the “torsion angles” is not convincing either, as the interface appears to be located in the upper parts of 5-HT2AR. Are changes in these upper parts of the helices (e.g., tilting) expected to be captured by the derived torsion angle, given the position of the angle measurement at the bottom of the 5-HT2AR?\nMoreover, while the authors mention in their rebuttal that they have provided uncropped gels/blots to allow further assessment of their cross-linking analyses within a Supplementary Data file, there actually is no such file within the documents shared for revision.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "In this revised study, the authors propose a heteromeric interface between 5-HT₂A and mGlu2 mediated by TM4–TM5 interaction and identify a novel allosteric mechanism through which the 5-HT₂A receptor modulates mGlu2 signaling. This conclusion is supported by an integrated approach combining TR-FRET, advanced computational modeling, cysteine crosslinking, and functional assays. While previous studies have examined the interaction between mGlu2 and 5-HT₂A, this work provides an elegant and mechanistically detailed advance, supporting how a class A GPCR can allosterically modulate mGlu2 dimer signaling through specific transmembrane interactions. The experiments are generally well controlled, leveraging the availability of existing tools and methods, and I believe that the authors have adequately addressed prior reviewers’ concerns by providing additional supporting data and reasonable explanations in their rebuttal. I have minor recommendations, which are outlined below.\nSpecifics:\nBecause allosteric modulation is probe-dependent, one would expect the FRET response of the mGlu2–5-HT2A complex to differ when agonists other than LY379268 are used. Was this assessed? In addition, how does increasing the expression of unlabeled 5-HT2A affect the intramolecular FRET signal of mGlu2 dimers? Such an effect would be anticipated, as the presence of 5-HT2A is expected to allosterically modulate mGlu2 dimer conformation and signaling . If these data have already been obtained, they should be explicitly mentioned and discussed, as they would substantiate the claims put forth.\nSuppl. Fig 5: It would be informative to provide the frequency (in percentage) for the common contact residues from TM4-TM5 of mGlu2 and 5-HT2A frequently observed in CGMD simulations.\nLine 198: “We quantified on blots the S5-HT2A monomer (M), dimer (D)198and S5-HT2A cross-linked to mGlu2 (mGlu2-5HT2A oligomers; O)…” this nomenclature should also be used in the figure.\nLine 226: “The above experiments indicate that TM4 of both receptors…” The description here lacks clarity regarding which receptors and complexes are being referred to. The authors should be more precise through the text (also mentioned in previous critics) in specifying whether these conclusions pertain to 5-HT2A–5-HT2A homomers or to the mGlu2–5-HT2A heteromer. This ambiguity is particularly confusing because the preceding results appear to describe 5-HT2A–5-HT2A interactions, whereas the subsequent experiment examines the mGlu2–5-HT2A complex. Clear and consistent identification of the receptor complexes under study is needed throughout the manuscript.\nLine 269: The authors must mean Suppl. Fig 17b-e.\nIn general, I recommend that the authors carefully revise the manuscript, potentially with editorial assistance, to improve clarity, flow, and consistency of tense, particularly in the Introduction. I highlight only a few representative examples from the Introduction; similar issues are also evident in the newly added text.\n“It is then of much interest to understand how one GPCR subtype can regulate the function of another ,as such information may be of interest to develop new drugs that precisely tune GPCR signaling.” I would suggest instead: It is therefore important to understand how one GPCR subtype regulates the function of another, as such knowledge may enable the development of drugs that precisely tune GPCR signaling?\n“In contrast, a weak FRET signal was obtained between SmGlu2 and the Halo-tagged M2 muscarinic receptor(N-terminal region of 18 residues), the HM2 construct used a negative control (Fig. 1b); and also sphingosine-1 phosphate receptor 1 (N-terminal region of 41 residues), the construct HS1P1122 (Supplementary Fig. 3).” The syntax and punctuation are odd.\n“It has also been studied for class C GPCRs, which classically form mandatory dimers, unlike all the other GPCRs, such as class A receptors, for which it has been demonstrated that the necessary and sufficient signaling unit is monomer”.\nAmbiguous sentence…What has been studied? Please consider rephrasing."}, "author_response": "Rebuttal point-by-point\nReviewer #1:\nThis is a very elegant manuscript with a massive amount of work testing the residues of the 5-\nHT2A and mGlu2 receptors that mediate their physical interaction as a GPCR heterocomplex. The\ntechniques involve TR-FRET, advanced computer modeling, cysteine crosslinking, and functional\nassays using both nanobody-based FRET and other traditional functional assays such as IP1. The\nauthors propose an heteromeric interface between 5-HT2A and mGlu2 mediated via an\nasymmetric TM4-TM5 interaction, as well as a new allosteric interaction via which 5-HT2A\nmodulates the G protein coupling preferences of the mGlu2 among Go vs Gi.\nWe wish to thank the referee for his/her very positive view of our study. As detailed below, we\ndid our best to clarify all the critics raised on our work.\nComments:\nThe authors use non-cell permeant fluorescent substrates to test FRET (or molecular proximity\nbetween 5-HT2A and mGlu2). However, other groups have shown that 5-HT2A has a high level\nof basal endocytosis and intracellular localization (PMID: 18703670, PMID: 33082287,\nPMID:36795823(Lopez-Gimenez et al., 2008; Toneatti et al., 2020; Vargas et al., 2023)). Does\nthe use of non-cell permeant FRET substrates limit the population of 5-HT2A-mGlu2 complexes\nthat is measured?\nTo clarify this point, we did examine the localization of either mGlu2 and 5-HT receptors fused\n2A\nto RFP and eGFP, respectively, in the cells used for our study. We compared our conditions with\nthe one described in these three references (Rebuttal-Table 1). In our study, in basal conditions\nthe rat 5-HT -eGFP receptor construct expressed alone is found mainly at the level of the plasma\n2A\nmembrane, but some intracellular 5-HT receptors can be observed (Rebuttal-Figure 1a). This\n2A\nis in contrast to these previous studies where there is a strong intracellular localization of the\nrecombinant human 5-HT receptor in basal conditions (Toneatti et al Sci Signal (2020) PMID\n2A\n33082287 ; Lopez-Gimenez et al Mol Pharm (2008) PMID 18703670 ; Vargas et al Science (2023)\nPMID 36795823). These differences with the previous studies might be due to the difference in\nthe constructs, including the signal peptide used in our study, or of the conditions of transfection\nand expression (Rebuttal-Table 1). But interestingly, when our rat 5-HT -eGFP construct is co-\n2A\ntransfected with the rat mGlu2-RFP construct, 5-HT receptors are no longer observed\n2A\nintracellularly (Rebuttal-Figure 1c), suggesting mGlu2 may help most of the 5-HT receptors to\n2A\nreach the cell surface.\nOf note, upon 5-HT agonist activation, a larger amount of intracellular 5-HT -eGFP receptors\n2A\ncan be detected when expressed alone (Rebuttal-Figure 1a) or co-transfected with mGluR2\n(Rebuttal-Figure 1c), with 5-HT receptors co-localized with mGlu2 in some of the intracellular\n2A\ncompartments. This suggests that some of the 5-HT receptors can co-internalize with mGlu2,\n2A\nconsistent with a fraction of these receptors being interacting.\nIn contrast, our rat mGlu2-RFP construct expressed alone is localized mostly at the plasma\nmembrane (Rebuttal-Figure 1b), and we did not observe detectable mGlu2 internalization upon\nagonist activation, in agreement with previous studies (Abreu et al., Cell Rep 2021 – PMID\n33910009; Cimadevila et al., Cell Rep 2025 – PMID 40748754).\nIn our FRET study, although we used different constructs (Halo-tagged rat 5-HT and SNAP-\n2A\ntagged rat mGluR2 constructs with unmodified C-terminal regions), we believe that under our\n1\nexperimental condition, most of the receptors are similarly and mostly at the cell surface, and\ntherefore, are prone to be labeled with the non-cell permeant SNAP and Halo substrates.\nHowever, such conclusions would need further experiments that are, we think, out of the scope of\nthe present study as they are not needed for our main conclusion.\nmGlu2 alone 5-HT alone mGlu2 + 5-HT Reference Remarks\n2A 2A\nLow High High for both Toneatti - Human cMyc-5-HT -\n2A\nmGlu2 and 5- (2020) mCherry or YFP\nHT 2A PMID - Flip-in HEK293\n33082287 inducible stable\ntransfection\n- Induced for 24-48 h\nN.D. High N.D. Lopez- - Human cMyc-5-HT -\n2A\nGimenez eCFP\n(2008) - Flip-in HEK293\nPMID inducible stable\n18703670 transfection\n- Induced for 24 h\nN.D. High N.D. Vargas - cMyc-5-HT -eCFP\n2A\n(2023) - HEK293T - 24h after\nPMID transient transfection\n36795823\nLow Low Low for both our - Halo or SNAP-tagged\npresent receptors\nstudy - Transient transfection for\n18 h in HEK293 cells\nRebuttal Table 1. Comparison of the levels of mGlu2 and 5-HT receptors intracellularly\n2A\nretained in the indicated basal conditions. N.D., not determined.\n2A\nin basal or agonist induced conditions. Fluorescence detection of 5-HT -eGFP and mGlu2-RFP in transfected\n2A\nHEK293 cells after incubation with control buffer, LY379268 (20 M) mGlu2 agonist or serotonin (5-HT, 10\nM) 5-HT agonist. Before treating cells with drugs, they were incubated for 4 h in Glutamax medium without\n2A\nserum and then starved with PBS for 0.5 h. Images were taken after a 30 min incubation with or without the\nindicated agonist.\nCan the authors provide a better explanation of the rationale behind the position of Halo-tag and\nSnap-tag? If the Halo-tag is located at the N-terminal of mGlu2, is the distance considering the\nlarge Venus flytrap extracellular domain of the class C GPCRs short enough to allow FRET with\nthe Snap-tag located also at the N-terminal of the 5-HT2A? The authors mention this at the\nbeginning of the results sections, but the effect of distance between the two N-terminals of mGlu2\nand 5-HT2A is not clearly described.\nIt is difficult to give a precise distance between the fluorophores attached to the Snap and Halo\ntags fused to the N-terminus of each receptor, as both are linked via a flexible linker (see Supp\nFig. 4 in Olofsson et al Nat Comm 2014 – PMID 25323157). It is true that the distance between\nthe N-terminus of mGlu2 (large extracellular domain of 563 amino-acids) and the surface of the\nplasma membrane is close to 9 nm, a distance quite large not expected to give a significant FRET\nwith the short N-terminus of 5-HT (68 amino-acids). This is why we attached the largest tag\n2A\n(Halo-tag; 33 kDa) to the N-terminus of 5-HT and the Snap-tag (20 kDa) to the N-terminus of\n2A\nmGluR2, to bring up the fluorophores attached to 5-HT , reaching a distance compatible with\n2A\nFRET between fluorophores.\nAs suggested by the reviewer, we compared the FRET signal between the two receptors when the\nHalo-tag fused to the N-terminus of mGlu2 was co-transfected with a 5-HT receptor fused with\n2A\nSnap tag at its N-terminus. A smaller FRET signal was measured (Rebuttal Figure 2) compared\nto the use of Snap-tagged mGluR2 co-transfected with Halo-tagged 5-HT (Figure 1b). In\n2A\naddition, we show that the FRET efficacy is dependent with the pair of fluorophores used. As\nexpected, using a pair with a smaller Ro (50 Å for Lumi4Tb and Green) give a smaller signal than\na pair with a larger Ro (58 Å for Lumi4Tb and Red; Scholler et al., Nat Chem Biol 2017 – PMID\n28135236). Finally, the large FRET signal used with the fluorophore pair Lumi4Tb and Green\n3\nshowed that the distance between the Snap-tag attached to mGlu2 and Halo-tag linked to 5-HT\n2A\nis within the 50 Å range (Figure 1b).\nRebuttal Figure 2: Intensity of the FRET signal at the cell surface between HmGlu2 and either S5-HT or SM2\n2A\nreceptors in cotransfected HEK293 cells, after colabeling with the non-cell permeant fluorophores Halo-Tb as\ndonor and either SNAP-Red or SNAP-Green as acceptors. The expression of HmGlu2 (Exp ) and S5-HT at\nH 2A\ncell surface was measured by the emission signal of Halo-Tb or Snap-Tb, after individual labeling of separate\ncell batches. The expression ratio of S5-HT / HmGlu2 and SM2 / HmGlu2 are is 1.24 and 1.66, respectively.\n2A\nData are mean ± SEM from a typical experiment performed three times (n=3).\nThese data are presented in the new Supplementary Figure 1 of the revised manuscript.\nWhy the ratios of ExpH/ExpS in Fig 1 have a shorter range (less that 2) for the SmGlu2-H5-HT2A\nexperiments, as compared to SmGlu2-HmGlu2 and SmGlu2-HM2R (above 3)?\nThis is due to a lower expression of the Halo-5-HT receptor, compared to Halo-mGlu2 or\n2A\nHalo-M2 receptors. This is now better stated in our revised manuscript.\n5-HT and M2) in three independent experiments. (a, d, g) FRET saturation analysis between SmGlu2 and\n2A\nHmGlu2, H5-HT or HM2 coexpressed at the cell surface of HEK293 cells as indicated. (b, e, h) Expression\n2A\nlevel of SmGlu2 at cell surface when coexpressed with HmGlu2, H5-HT or HM2. (c, f, i) Expression level of\n2A\nHmGlu2, H5-HT or HM2 at cell surface when co-expressed with SmGlu2. A fixed amount of SmGlu2 (20 ng)\n2A\nwas cotransfected with increased amounts (from 0 to 80 ng) of HmGlu2, H5-HT or HM2, as indicated.\n2A\nExpression levels were normalized to SmGlu2 expressed alone in the positive control (SmGlu2 + HmGlu2).\nExperiments 1, 2 and 3 are shown in (a-c), (d-f) and (g-i), respectively.\nThese data are presented in the new Supplementary Figure 2 of the revised manuscript.\nStatistical analysis (comparing FRETmax and FRET50) are not assessed in the FRET saturation\nassays.\nWe have plotted these values from the three independent TR-FRET saturation experiments above.\nThese data were analyzed in one-site specific binding. We can provide the values of Bmax and K\nD\nwhich are similar to FRET Max or FRET respectively (See Rebuttal Table 2). We found a\n50\nsimilar K values for the different receptors, but the estimated Bmax values are statistically\nD\ndifferent.\nRebuttal Table 2: Summary of the Best fit values of Bmax and K for three independent TR-\nD\nFRET saturation experiments.\nN=1 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.06 2.00 0.90\nK 0.48 0.73 0.51\nD\nN=2 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.17 1.22 0.49\nK 0.75 0.22 0.43\nD\nN=3 SmGlu2 + HmGlu2 SmGlu2 + H5-HT SmGlu2 + HM2\n2A\nBmax 3.52 2.53 0.98\nK 0.57 0.88 0.76\nD\nAuthors show FRET signal using the “wild-type” 5-HT2A and mGlu2 receptors in Fig 1. This\nreplicates what others have shown using assays in HEK293 cells under a “basal” state (i.e., without\n5-HT2A or mGlu2 agonists) (PMID: 18297054, PMID: 19590495, PMID: 22300836; PMID:\n29858599(Gonzalez-Maeso et al., 2008; Rives et al., 2009; Delille et al., 2012; Murat et al., 2019)).\nHowever, the authors use a constitutively active mutant (mGlu2-Y6.58C, or mGlu2-Ctrl) to\nconduct all the very elegant cysteine-crosslinking studies. Have the authors tested cysteine-\ncrosslinking with the “wild-type” mGlu2?\nThis is important because as mentioned (and demonstrated) by authors, the structural arrangement\nof the mGlu2 homodimer is changed upon agonist activation. Previous work by others indicates\nthat TM4 (and in particular three alanine residues located at the intracellular half of the TM4 of\nthe mGlu2: Ala4.40, Ala4.44 and Ala4.48) are necessary for the mGlu2 to form a GPCR complex\nwith 5-HT2A (PMID: 23129762; PMID: 18297054; PMID: 37989895(Gonzalez-Maeso et al.,\n2008; Moreno et al., 2012; Gao et al., 2024)). This has been validated using photoactivatable\nunnatural amino acids (PMID: 32726588(Shah et al., 2020)). How can the authors integrate their\nfindings with a constitutively active mGlu2 mutant and previous findings using the wild-type\nmGluR2?\nOur data highlight the fact that 5-HT receptor preferentially interact with the active forms of\n2A\nmGlu2, a conformation that exposes the TM4-5 interface (PDB 7MTS) where the 5-HT\n2A\ninteracts. Accordingly, WT mGlu2 and 5-HT can indeed interact, as illustrated in Figure 1b.\n2A\nWhen using Cys-crosslinking experiments, we did test the WT mGlu2 and observe a weak cross-\nlinking (this was indeed shown in Supplementary Fig 3c,d,f in our original paper; see below as\nRebuttal Figure 4). We interpret this as a difficulty of the two proteins to orient correctly due to\nthe structural dynamics of the mGlu2 receptor likely oscillating from the inactive to the active\nstate, limiting the possibility of a disulfide bound formation. This is the reason why we used a\nmGlu2 receptor mutant stabilized in its active state, making the TM4-5 always available for\ninteraction with the 5-HT receptor. Under such condition, a higher crosslinking could be\n2A\nobserved (New Figure 3g-h). This is perfectly consistent with our model.\nRebuttal Figure 4. (a) Cross-linking of the indicated constructs of S5-HT mutant and HmGlu2 wild-type or\n2A\nmutant after labeling with non-cell permeable fluorescent Snap-Alexa Fluor 782 substrates. The mutant\nconstructs contain a single cysteine at positions mGlu25.42 and 5-HT 5.41. The results were obtained after pre-\n2A\nincubation with mGlu2 receptor agonist LY379268 and CuP. (b) Change of the percentage of cross-linked\nmGlu2-5-HT hetero-oligomers at the cell surface in the presence or absence of LY379268 (10 M). The\n2A\namount of S5-HT subunits labeled at the cell surface by the Snap-Surface Alexa 782 substrate was quantified\n2A\nby emission at 800 nm. Data are mean ± SEM from at least three independent experiments. One-way ANOVA\nwith Dunnett’s multiple comparisons test with ***P < 0.001 and ns, not significant. (c) SDS-PAGE analysis of\nthe mGlu2-5-HT hetero-oligomer. Detection of the indicated co-expressed S5-HT and HmGlu2 wild-type or\n2A 2A\nmutants after labeling with non-cell permeable fluorescent substrates. Snap-tag and Halo-tag were labeled with\nSnap-Alexa Fluor 782 and Halo-Alexa Fluor 660, respectively. The cross-linked mGlu2-5-HT complex can\n2A\nbe specifically detected after introducing cysteine mutations in 5.41 of 5-HT and 5.42 of mGlu2.\n2A\nPanels (a) and (b) are presented in the New Figure 3 of the revised manuscript, and panel (c) is\npresented in the Supplementary Figure 10 of the revised manuscript.\n6\n5-HT2A also forms homocomplexes (as shown by authors in Figure S4 and by others in PMID:\n19279328(Brea et al., 2009b)). Would it be possible to model an alternative arrangement of the\nGPCR protomers in Fig 4D, showing proximity between the two 5-HT2A protomers?\nIn our cross-linking experiments, two conformational arrangements for the 5-HT homodimer\n2A\nwere found involving either TM4-5 or TM1 interfaces (Supplementary Figure 14). These results\nagree with the study by Brea et al. (Brea et al. (2009) Mol Pharm – PMID 19279328), who\npresented data supporting the existence of 5-HT receptor homodimers in live cells by\n2A\ncoimmunoprecipitation studies and FRET experiments. Interestingly, they proposed a\nmathematical model consisting of a three-state preformed homodimeric receptor, including an\ninactive state, R2, and two active states, R2* (inositol phosphate signaling pathway) and R2**\n(arachidonic acid signaling pathway), where the active receptor states were selectively\ndistinguished by typical and atypical antipsychotics. It is worth noting that, as the authors\nacknowledged, their data were also compatible with a dynamic equilibrium between monomeric\nand dimeric 5-HT receptors showing different functionality (i.e., monomeric receptors signaling\n2A\nvia the inositol pathway and dimeric receptors signaling via the arachidonic acid pathway, the\nlatter displaying ligand-dependent cooperativity).\nWe appreciate the suggestion of the reviewer, and we have modeled a receptor complex consisting\nof the mGlu2 obligate homodimer and a 5-HT homodimer to fit Figure 4d of our manuscript.\n2A\nOf the two conformational arrangements provided by our crosslinking experiments for the 5-HT\n2A\nhomodimer, only the one featuring the TM1-TM1 interface is consistent with the proposed mGlu2-\n5-HT heteromeric model (Rebuttal Figure 5). As such, a 5-HT dimer composed of subunits\n2A 2A\ninteracting via TM4-5, are unlikely able to interact with mGlu2. It is however our view that the 5-\nHT receptor dimers are likely dynamics, oscillating between a monomeric and a dimeric state,\n2A\nthen making possible the interaction of the 5-HT receptor with the active form of mGlu2\n2A\nhomodimer.\nRebuttal Figure 5. mGlu2-5-HT heteromeric receptor model including a 5-HT homodimer. The TM1-\n2A 2A\nTM1 interface for the 5-HT homodimer is compatible with the heteromerization interface in mGlu2-5-HT\n2A 2A\ncomplex based on the cross-linking results.\nThese data are presented in the new Supplementary Figure 14 of the revised manuscript.\nThese studies are only tangentially related to the model the authors are proposing.\nWe agree with the referee, but because a few laboratories questioned the veracity of the previously\nreported data coming from a unique laboratory, we still think it was important to confirm this\ninteraction using different approaches. Validating these proposals was indeed essential to reach\nour aim that 5-HT receptors could potentially activate mGlu2 by interacting with an interface\n2A\nonly accessible in the active form of the receptor. Of importance, our data are the first to\ndemonstrate the conformational dependence of the mGlu2-5-HT receptor interaction. In our\n2A\nview, this is a very important and novel finding.\nThe crosslinking studies provide data that are consistent with the structural model, and the amount\nof work represented by these studies is appreciated. However, the results ultimately show that a\nTM4/5 interface for mGlu2 becomes accessible for crosslinking when this receptor is active, which\ncould have been inferred from the already-available structures. The information gained here isn’t\nleveraged for functional studies. Again, the crosslinking studies aren’t controlled by any other\nclass A GPCR.\nThe experiments were designed and done before the mGlu2 structures were available, and based\non our hypothesis for mGlu2 activation (Xue et al., NCB 2015 – PMID 25503927), a model that\nwas not accepted by many groups as the intersubunit movement was judged too big. Since then,\nour proposal has been validated by the cryoEM structures of mGlu2 in various states. Although\nthe mGlu inactive state structures show 7TM subunit often facing each other via TM4-5, this is\nnot always the case (see mGlu3 inactive states in Fang et al., Cell Res 2022 – PMID 35236939),\nand this very static view is unlikely real, as the 7TMs are likely to move one compared to the other,\ngiving some possible flexibility for a class A GPCR to interact with the TM4-5 interface. Of note,\nthe very different 7TM relative positions in the various mGlu inactive state structures (see\nstructures of mGlu1, 2, 3 and 5 in the inactive state; also reviewed by Liu et al (2023) Am J Physiol\nCell Physiol – PMID 37184233) support such a constant dynamic of the two 7TM domains in the\ninactive conformation.\nThe reviewer asked about the crosslinking studies by any other class A GPCR. We believe that the\nbest negative control for the relevant crosslinking are the absence of cross-linked observed\nbetween mGlu2 TM4-5 and TM1 and TM6 of 5-HT receptor (because TM1 is also involved in\n2A\nhomodimer interface) (see Supplementary Figure 11).\nGiven the long history of this heterodimer (first described in 2008) the saturation TR-FRET studies\n32\ncover old ground using methods with well-known technical limitations. A “saturating” curve is\nuninterpretable if acceptor density is normalized using donor density, and changes in response to\nagonist (as in Figure 3a) could reflect conformation as much as association. Agonist-induced\nchanges are not controlled by any other TM protein. Here again there are unexplained\ninconsistencies. In the authors’ model some 5HT2a interacts with unliganded mGlu2 because of\nconstitutive activity or ambient glutamate. Why then does agonist apparently enhance\n“association” with 5HT2a (Fig. 3a), but antagonist does not do the opposite (S2b).\nRegarding the original Fig 3a, the referee is correct, this can come either from an increased mGlu2-\n5-HT complexes, but this can also come from a slight change in distance between the\n2A\nfluorophores due to the conformational change in the VFTs. This is now clearly mentioned in the\nResults section (page 8), and used as an argument to directly test the agonist dependency of the\ninteraction using cross-linking experiments.\nIn the new Figure 3, we now include the essential data that were originally shown in Supp Fig\n3c,d, showing a very low cross-linking in the absence of mGlu2 agonist, while a large amount of\ncrosslinked receptors are observed in the presence of the mGlu2 agonist. It is likely that the\ncrosslinking occurs when the interaction is stable enough to allow the two thiols groups to find the\ntime to be in close proximity and in the correct orientation for the disulfide bond to form. However,\nshorter transient interaction between mGlu2 and 5-HT could occur thanks to the constant\n2A\ndynamics of the mGlu2 7TM dimer that likely exists when both VFTs are stabilized open with an\nantagonist. Indeed, although the TM domains are well constraint and always found to interact via\nTM6 in all active state class C GPCRs, the relative position of the two 7TMs are very variable in\nthe apo or antagonist bound states (see structures of mGlu1, 2, 3 and 5 in the inactive state; also\nreviewed by Liu et al (2023) Am J Physiol Cell Physiol – PMID 37184233). It is therefore not\ntotally surprising that the basal FRET observed in the original Supp Fig 2b is not inhibited by\nLY341495. This point is now discussed in the revised manuscript.\nThe functional studies rely on a defective 5HT2a mutant F173P, which is described as a mutation\nin the intracellular part of TM3 that blocks G protein coupling. The rat 5HT2a sequence has no F\nresidues in the intracellular part of TM3, and residue 173 is the R in the middle of the DRY motif.\nNo citation is provided for whatever this mutant is, and the methods section on plasmid\nconstruction is also unhelpful. When used it is not clear than this mutant receptor was expressed\nat the cell surface, and validation for loss of function is minimal. It is not clear that Gqi9 is present\nin Figure 5e, which is necessary to interpret Figure 5F.\nWe are very sorry for this mistake, yes, position 173 in the 5-HT receptor corresponds to the R\n2A\nof the DRY motif. This mistake has been corrected in the entire manuscript, as well as in every\nfigures and Supp figures. This R residue is highly conserved in class A GPCRs (Weis & Kobilka\nAnn Rev Biochem (2018) – PMID 29925258; Rovati et al (2007) Mol Pharm – PMID 17192495).\nAlthough this mutation of R173 into P has not been reported to our knowledge, mutation of this R\nresidue has been studied for many class A GPCRs such as the angiotensin AT1 receptor\n(Suonivuori et al Lefkowitz (2020) Science). How this mutant has been generated is now better\nexplained in the Methods section (Page 20). We also refer to other studies in which mutation of\nthis R residue from the DRY motif suppresses G protein signaling (Rovati et al (2007) Mol Pharm\n– PMID 17192495).\nThis mutation prevents G protein activation by this receptor, as well illustrated in the original Supp\nFig 9c using the G protein biosensors, and in the original Fig 5e, when IP production is measured,\n1\neven in the presence of over-expressed Gqi . As indicated below, this mutant receptor expresses\n9\nvery well at the cell surface (Rebuttal Figure 19), an information now added in the revised paper.\n80000\ne\nc\na\nfr 60000\nu\ns\nlle\nc\nta 40000\nn\no\nis\ns\ne 20000\nr\np\nx\nE\nP\nS m H5- T2 A\n+ H\nGl\nu 2\n+\nH5-\nS m u 2\nGl\nm\nS\nRebuttal Figure 19. Expression level of H5-HT wild-type and R173P mutant at the cell surface in the\n2A\nFigure 5d. The amounts of H5-HT and H5-HT R173P at cell surface were measured by the emission signal of\n2A 2A\nHalo-Tb. H5-HT R173P mutant showed similar expression level to wild-type H5-HT in presence of co-\n2A 2A\ntransfected SmGlu2. Data are mean ± SEM from a typical experiment performed three times (n=3).\nWe now include additional experiments with this mutant, looking at the effect of mGlu2 on the\nactivity of the 5-HT receptor, looking at its capacity to generate a calcium signal (see Rebuttal\n2A\nFigure 6, page 10 of this rebuttal). We show that indeed, when 5-HT receptors are co-expressed\n2A\nwith mGlu2, the mGlu2 agonist LY379268 generates a calcium signal, an effect not observed with\nthe R173P mutant (data are show in our answer to referee #1 in Rebuttal Figure 6, page 10 of\nthis rebuttal). And, the original Fig. 5e-f have been modified to clarify that Gqi has been used in\n9\nthe experiments illustrated in panels e and g in the new Figure 5.\nThroughout the manuscript there are instances of questionable experimental rigor. For example,\nin Figure 1 we are told “Data are means ± SEM of triplicates from a typical experiment”,\nwhich suggests three technical and one biological replicate. Similar descriptions appear in Figures\n3a, 5b, S2, etc.. The legend for Figure 6b says 3 independent experiments but there are 10-12 data\npoints on the bars, suggesting the authors are plotting technical replicates. Since an ANOVA is\nperformed on these data it’s not clear what they are including. In Fig. 6c there are 6 points on the\ngraph for “a typical experiment performed at least 3 times”.\nWe clarified the Fig. 1b, 3a, 5b, Supp Fig 2a by indicating that the data shown are means +/- SEM\nare from three independent experiments (n=3).\nFor Fig. 6b, six independent experiments have been performed for LY37 and buffer conditions,\nwhen mGlu2 is expressed alone or coexpressed with 5-HT mutant. Similarly, for Fig. 6c, six\n2A\nindependent experiments have been performed for LY37 and buffer conditions, when mGlu2 is\nexpressed alone or coexpressed with 5-HT mutant.\n2A\nWe now carefully checked that the number or replicates and number of independent experiments\nare correctly mentioned in every figure legends.\ndiscussion to place their findings in context of what is already known. For example, how can these\nobservations be reconciled with a report that mGlu2-dependent activation of Gi/o is unchanged in\n5HT2a -/- mice (PMID: 26758213(Moreno et al., 2016)).\nWe tried to improve our discussion, respecting the size limit, better referring to the previous\npublished work, done almost exclusively by the group of Javier Gonzales-Maeso. Indeed, we do\nthink it was important to confirm his data using eventually different approaches, as these were\nessential to validate our hypothesis that 5-HT receptor would stabilize the active conformation\n2A\nof the mGlu2 dimer. Our aim was not to go further into the physiological meaning, as these were\nlargely discussed in relation to the antipsychotic effects of mGlu2 and 5-HT receptor ligands by\n2A\nJavier. Regarding the specific point raised by the referee: even though we do believe the 5-HT\n2A\nreceptor can indeed regulate the activity of mGlu2, such a regulation may occur in some specific\nbrain area or neuronal subdomains, such that only a very small fraction of the mGlu2 receptors\ncan be regulated by 5-HT receptors. This is expected as most 5HT receptors are post-synaptic,\n2A 2A\nwhile the mGlu2 receptors are pre-synaptic. In addition, we do believe this effect is transient,\nsuch that even for mGlu2 receptors that can be regulated by 5-HT , only a fraction of these will be\n2A\nassociated with 5-HT at a given time. Taking these points together, one is not expecting that the\n2A\ndeletion of 5-HT receptor will significantly affect the capacity of mGlu2 to regulate Gi/o\n2A\nproteins. This does not exclude an important role of this regulation occurring at specific sites\nimportant for the antipsychotic effects of mGlu2 or 5HT ligands.\n2A\n35"}]

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