# Non-canonical internalization mechanisms of mGlu receptors

Marta Cimadevila, Junke Liu, Damien Maurel, Isabelle Brabet, Marine Hoscar, Julia Drube, Carsten Hoffmann, Asuka Inoue, Philippe Rondard, Pierre-André Lafon, et al.

► To cite this version:

Marta Cimadevila, Junke Liu, Damien Maurel, Isabelle Brabet, Marine Hoscar, et al. Non-canonical internalization mechanisms of mGlu receptors. Cell Reports, 2025, 44 (8), pp.116068. 10.1016/j CELREP.2025.116068. hal-05213663

HAL Id: hal-05213663

https://hal.science/hal-05213663v1

Submitted on 18 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
**Cell Reports**

# Non-canonical internalization mechanisms of mGlu receptors

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Marta Cimadevila, Junke Liu,
Damien Maurel, ..., Pierre-André Lafon,
Laurent Prézeau, Jean-Philippe Pin

## Correspondence

jean-philippe.pin@igf.cnrs.fr

## In brief

Using DERET to analyze mGlu receptor internalization kinetics, Cimadevila et al. demonstrate that only mGlu1, mGlu5, mGlu3, and mGlu2-3 dimers undergo agonist-induced internalization even in the absence of β-arrestins or GRKs. However, β-arrestins emerge as key players in constitutive internalization of mGlu receptors.

## Highlights

* Only mGlu1, mGlu5, mGlu3, and mGlu2-3 undergo agonist-induced internalization

* Internalization still occurs in the absence of GRKs that tune this process

* β-arrestins are not required for agonist-induced internalization of mGlu receptors

* β-arrestins are required for constitutive internalization only

<figure><img src="image_3.png" /></figure>
Article

# Non-canonical internalization mechanisms of mGlu receptors

Marta Cimadevila,<fnref n="1" /> Junke Liu,<fnref n="1" /> Damien Maurel,<fnref n="1" /> Isabelle Brabet,<fnref n="1" /> Marine Hoscar,<fnref n="1" /> Julia Drube,<fnref n="2" /> Carsten Hoffmann,<fnref n="2" /> Asuka Inoue,<fnref n="3" /> Philippe Rondard,<fnref n="1" /> Pierre-André Lafon,<fnref n="1" /> Laurent Prézeau,<fnref n="1" />,<fnref n="4" /> and Jean-Philippe Pin<fnref n="1" />,<fnref n="4" />,<fnref n="5" />,*

## SUMMARY

Cell surface G protein-coupled receptors (GPCRs) are tightly regulated through constitutive and agonist-induced internalization. While the mechanisms of constitutive internalization remain elusive, agonist-induced internalization very often involves receptor phosphorylation by GPCR kinases (GRKs) and β-arrestin recruitment. Dimeric class C metabotropic glutamate (mGlu1–mGlu8) receptors regulate synaptic transmission, but their internalization process remains ambiguous. Here, using diffusion-enhanced energy transfer (DERET), we show that mGlu receptors undergo constitutive internalization that mostly relies on β-arrestins. However, only mGlu1, mGlu5, and mGlu3 homodimers and mGlu2-3 heterodimers undergo agonist-induced internalization. This process depends on protein adaptor complex 2 (AP2) but still occurs in the absence of GRKs or β-arrestins, with the latter acting as fine-tuners. This systematic study further highlights how class C receptors differ from other GPCRs, revealing non-canonical internalization mechanisms that remain to be characterized. These insights into mGlu receptor dynamics will contribute to the development of therapeutic strategies targeting mGlu receptors.

## INTRODUCTION

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that serve as the targets for approximately 30% of drugs on the market.<fnref n="1" /> Although primarily embedded in the cell membrane, they are highly dynamic and can internalize into intracellular compartments. This process can occur in the absence of ligands (constitutive internalization) or upon agonist binding (agonist-induced internalization), potentially via distinct mechanisms.

Agonist-induced internalization is canonically described as a clathrin-mediated process,<fnref n="2" /> although other mechanisms, including caveola-mediated pathways,<fnref n="3" /> have also been reported. Upon agonist binding, GPCRs are phosphorylated by GPCR kinases (GRKs), leading to β-arrestin (βarr) recruitment. Consequently, βarrs are activated and function as cargo adaptors for the protein adaptor complex 2 (AP2). AP2 drives the invagination of the activated GPCR in clathrin-coated pits.<fnref n="4" /> In contrast, the mechanisms underlying constitutive GPCR internalization remain poorly understood and are largely described as non-clathrin mediated.<fnref n="3" />,<fnref n="5" />,⁶

For class C GPCRs, such as metabotropic glutamate (mGlu) receptors, multiple studies have investigated their desensitization mechanisms.⁷,⁸ However, their internalization processes

remain largely unknown and controversial. While some studies suggest that certain mGlu receptors follow the canonical internalization pathway,⁹⁻¹¹ others indicate that mGlu receptors internalize via a clathrin-independent mechanism.¹²⁻¹⁴ Understanding these pathways is crucial, as mGlu receptors are promising drug targets for chronic neurological disorders, including Parkinson's disease, schizophrenia, and Alzheimer's disease.¹⁵ However, drug candidates targeting mGlu receptors have failed in clinical trials, primarily due to a lack of efficacy.¹⁶ Since long-term treatments depend on sustained target availability, receptor internalization might compromise the chronic effects of drugs that activate mGlu receptors.

Therefore, we aimed to precisely determine the internalization profiles and mechanisms of mGlu receptor internalization. We employed diffusion-enhanced resonance energy transfer (DERET) combined with protein knockout or downregulation. We show that mGlu receptors internalize through a non-canonical mechanism despite involving classic internalization components. Upon agonist binding, mGlu1, mGlu5, and mGlu3 internalize in a βarr-independent manner that requires AP2. In contrast, constitutive internalization depends on βarr for most of them and is precisely modulated by GRKs. These findings reveal that mGlu receptors use a unique internalization machinery that remains to be fully characterized,

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Institute of Functional Genomics, University of Montpellier, CNRS, INSERM, 34094 Montpellier, France</footnote>

<footnote n="2">²Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, 07745 Jena, Germany</footnote>

<footnote n="3">³Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan</footnote>

<footnote n="4">⁴These authors contributed equally</footnote>

<footnote n="5">⁵Lead contact</footnote>

<footnote>*Correspondence: jean-philippe.pin@igf.cnrs.fr</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2025.116068</footnote>
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

D

<figure><img src="image_8.png" /><figcaption>**Figure 1. DERET revealed internalization properties of ST-mGlu5**</figcaption></figure>

(A) Schematic of the experimental approach used to measure internalization kinetics by DERET. Red circles, agonist; black circles, Lumi4-Tb when it is quenched; yellow circles, Lumi4-Tb when emitting light.

(B) Internalization kinetics of ST-mGlu5 in the absence of agonist (vehicle), in the presence of 100 µM glutamate (+GLU) or in the presence of 100 µM LY341495 (+LY34).

(C) Agonist-induced internalization of ST-mGlu5 measured after 60 min in the presence of increasing doses of glutamate (+GLU), quisqualate (+QUIS), LY341495 (+LY34) or LY341495 (+100 nM quisqualate). Data are normalized to glutamate-induced internalization after 60 min of incubation at 37°C.

(D) Agonist-induced internalization (1 µM quisqualate, represented as red circles) of the CT-mGlu5 subunit when co-expressed with the ST-mGlu5Y subunit (green line and green stars) and the ST-mGlu5Y subunit when co-expressed with the CT-mGlu5 subunit (blue line and blue stars). Data are normalized to glutamate-induced internalization of mGlu5-only expressing cells.

Data are means ± SEM of at least three independent experiments performed in triplicates.

providing fundamental insights that could impact therapeutic strategies.

## RESULTS

### Identification of mGlu5 internalization kinetics

Internalization kinetics have been measured by DERET, an approach that takes advantage of the quenching of the long-life emission of lanthanides by diffusible acceptors at high concentration. GPCRs at the plasma membrane are covalently labeled with a lanthanide through a suicide enzyme genetically introduced at their N termini. Unless otherwise noted, constructs were covalently labeled using the SNAP-tag enzyme, a self-labelling protein tag that will covalently bind to benzylguanine (SNAP) derivatives. Specifically, SNAP-Lumi4-Tb (Lumi4-Tb) was used as substrate. Then, the addition of a saturating concentration of fluorescein quenches the fluorescence of the lanthanide.¹⁷,¹⁸ If the GPCR is internalized, then the lanthanide is physically separated from the quencher, allowing its fluorescence to be recovered, and can be recorded over time to follow the internalization kinetics (Figure 1A).

Accordingly, ST-mGlu5 was labeled with SNAP-Lumi4-Tb (1 h at 4°C) at the plasma membrane (Table S1). Internalization was then started by addition of the indicated ligands and fluorescein and followed for 60 min at 37°C. Data were corrected by receptor expression as explained in the STAR Methods and fitted to previously described pharmacokinetic equations.¹⁹

ST-mGlu5 was constitutively internalized, as an increase in Lumi4-Tb luminescence was observed in the absence of agonist (Figure 1B, vehicle). Such an effect is not due to the possible action of ambient glutamate in the medium up to 45 min, as internalization is not inhibited by the competitive antagonist LY341495 (Figure 1B). The endogenous agonist glutamate was found to increase internalization (Figure 1B) in a concentration-dependent manner (Figure 1C), the same as for the selective agonist quisqualate (Figure 1C), with half-maximal effective concentrations (EC₅₀) values similar to those found in the literature²⁰ (Table S2). The effect of quisqualate is inhibited by the antagonist LY341495 with the expected potency (Figure 1C; Table S2). Note that LY341495 has the tendency to decrease the basal mGlu5 internalization after 60 min of incubation at 37°C, an effect that varies from experiment to experiment, likely due to variable levels of ambient glutamate accumulation in the assay medium.²¹ Accordingly, basal internalization was always measured in the presence of this antagonist, which acts on all mGlu receptors, to prevent any possible effect of ambient glutamate.

As mGlu receptors are mandatory dimers, we wondered whether both subunits need to be activated for internalization.²² To investigate it, we used a mutated subunit that is unable to bind glutamate, ST-mGlu5Y.²³ The co-expression of this subunit together with a CLIP-tagged version of mGlu5, CLIP-mGlu5 (CT-mGlu5), results in three populations of dimers: mGlu5-5, mGlu5Y-5Y, and mGlu5-5Y. Taking advantage of the two different suicide enzymes that tag mGlu5 and mGlu5Y (CT and ST, respectively), we could detect these three types
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_11.png" /></figure>

of dimers at the cell surface by time-resolved fluorescence resonance energy transfer (TR-FRET; Table S1). We could then selectively follow internalization of one subunit or the other by using the CLIP or SNAP tag and so evaluate the internalization properties of each dimer. We found that ST-mGlu5Y was unable to undergo agonist-induced internalization when co-expressed with CT-mGlu5 (Figure 1D). Also, agonist-induced internalization of the CT-mGlu5 subunit is decreased when increasing expression of mGlu5Y, and then the population of mGlu5-5Y heterodimers (Figure S1).

<caption>Figure 2. Kinetic analysis reveals different internalization profiles of mGlu homodimers</caption>

(A) Internalization profiles of group I mGlu receptors (ST-mGlu1 and ST-mGlu5) in the absence of agonist (basal, 100 µM LY341495) or 10 µM quisqualate (+Ago).

(B) Internalization profile of group II mGlu receptors (ST-mGlu2 and ST-mGlu3) in the absence of agonist (basal, 100 µM LY341495) or 10 µM LY354740 (+Ago).

(C) Internalization profile of group III mGlu receptors (ST-mGlu4, ST-mGlu6, ST-mGlu7, and ST-mGlu8) in the absence of agonist (basal, 100 µM LY341495) or 10 µM L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) for mGlu4, mGlu6, and mGlu8 and 10 µM LSP2-9166 for mGlu7 (+Ago).

Stacked bars represent constitutive internalization (const; given by the basal signal, black) and agonist-induced internalization (ago; calculated by subtracting basal signal, red) for each ST-mGlu receptor after 60 min of internalization at 37°C. Data represent the means ± SEM of at least three independent experiments performed in triplicates.

As CT-mGlu5 is the only subunit able to undergo agonist-induced internalization, these data indicate that CT-mGlu5 cannot drive ST-mGlu5Y in endosomes, demonstrating that the mGlu5-5Y dimer cannot undergo agonist-induced internalization. Agonist-induced internalization of mGlu5 requires both subunits to be in the active conformation.

Only three of eight homodimers undergo agonist-induced internalization

The eight mGlu subtypes are classified into three groups according to their sequence homology. Group I includes subtypes 1 and 5, group II includes subtypes 2 and 3, and group III includes subtypes 4, 6, 7, and 8. We explored the internalization profile of all mGlu receptors using their ST version (Table S1). All homodimers were constitutively internalized, as revealed by the increase in Lumi4-Tb

signal over time in the absence of agonist (Figure S2A, vehicle) and even in the presence of antagonist (Figure 2, basal). Specifically, mGlu5 and mGlu3 constitutive internalization reached a plateau after 20 min at 37°C.

However, none of the group III receptor basal internalization was increased with a saturating dose of agonist (Figures 2C and S2A). Specifically, among the eight mGlu homodimers, only three showed agonist-induced internalization: both group-I ST-mGlu1 and ST-mGlu5 and the group II mGlu3 receptors (Figures 2 and S2A). Surprisingly, upon agonist activation,
no ST-mGlu3 internalization could be detected within the first 5 min in the HEK293 cells used, meaning that, in this time lapse, constitutive internalization is blunted (Figure 2B). This delay was systematically observed in HEK293 cells (Figures 4C, 5C, and 7B) but not in other cell lines (see below). Certainly, addition of an agonist increased internalization over basal until the equilibrium, which is reached at around 30 min of agonist incubation. Compensatory mechanisms, such as recycling, could explain these kinetic profiles.

## Agonist-induced internalized receptor undergoes recycling

Recycling refers to the process where a GPCR located in the plasma membrane and then internalized comes back to the surface.²⁴ To evaluate mGlu receptor recycling, labeled ST-mGlu5 and ST-mGlu3 were incubated with an agonist for 60 min at 37°C to induce their internalization. Then, either the excess of agonist was removed, or the antagonist LY341495 was added, as antagonists have been reported to accelerate recycling.²⁵ If the fluorescent internalized receptor is recycled back to the plasma membrane, then its Lumi4-Tb will be quenched at the surface (Figures S3A and S3B), leading to a decrease in the amount of fluorescence.

Indeed, ST-mGlu5 was recycled back to the plasma membrane over time, and the addition of LY341495 did not significantly increase recycling when compared with only washing the agonist (Figure S3C). Similarly, ST-mGlu3 recycling was observed upon LY341495 antagonist application (Figure S3C), in contrast to the washing condition alone. We cannot exclude the possibility that, after washing, ambient glutamate is accumulating over time and reaches the concentration needed to activate mGlu3 due to its high glutamate affinity.²¹ The identification of receptor recycling after agonist-induced internalization highlights the relevance of measuring kinetics while evaluating mGlu receptor internalization.

## Only mGlu2-3 among the mGlu3-containing heterodimers internalizes upon agonist stimulation

It is now well accepted that mGlu receptors exist not only as homodimers but also as heterodimers with specific subunit composition.²²,²⁶ While group I mGlu receptors only form heterodimers between themselves, both group II and group III can assemble into any possible dimer combination. Among them, only the mGlu3 homodimer was found to undergo agonist-induced internalization. We then examined whether mGlu3-containing heterodimers could undergo agonist-induced internalization.

By co-expressing ST-mGlu3 with other mGlu receptor subtypes labelled with a different suicide enzyme, in this case an Halo-tag (HT), we were able to only label the non-agonist-induced internalizing subunit of the heterodimer with a Halo-Lumi4-Tb substrate (HT-mGlu2, HT-mGlu4, HT-mGlu7, and HT-mGlu8), and follow its internalization over time (Figure 3A). We did not consider the investigation of mGlu3-6. as mGlu6 is confined in the ON bipolar cells of the retina.²⁷ We first verified that heterodimers could be detected at the cell surface, at a different extent, using TR-FRET compatible substrates for SNAP and Halo (Figure 3B). Intriguingly, only the HT-mGlu2 sub-

unit underwent agonist-induced internalization in the presence of mGlu3 (Figure 3C), but not HT-mGlu4, HT-mGlu7, or HT-mGlu8 (Figure 3D). This indicates that mGlu2-3 is the only mGlu3-containing heterodimer to undergo agonist-induced internalization.

## GRKs are not mandatory for agonist-induced internalization

Three main molecular players are involved in the canonical GPCR internalization process: GRKs, βarr, and AP2. GRKs are kinases that can be dispersed in the cytoplasm (GRK2 and GRK3) or associated with the plasma membrane (GRK5 and GRK6). They phosphorylate the C-terminal domain of agonist-bound GPCRs as a first step in the internalization process for most GPCRs (Figure 4A), such as μ-opioid receptors (MORs).²⁸ Certainly, inhibition of GRK2/3 with the GRK inhibitor Cmpd101 decreased agonist-induced internalization of ST-MOR without affecting constitutive internalization (Figure 4B). The complete deletion of GRKs (ΔQ-GRK cell line²⁹ [ΔGRK]) blunted agonist-induced internalization of ST-MOR, internalization that could be rescued by overexpression of any of the GRKs (Figure 4B), validating our approach. In contrast, MOR constitutive internalization was independent of GRK expression (Figure 4B).

With this positive control in place, we then unveiled the role of GRKs in both constitutive and agonist-induced internalization of mGlu3, as this receptor undergoes both constitutive and agonist-induced internalization. Cmpd101 failed to induce a change of ST-mGlu3 internalization, both constitutive and agonist induced (Figure 4C). To completely evaluate the role of GRKs, we transiently expressed the ST-mGlu receptors in ΔGRK cells and in parental cells (Table S1). Even though receptor expression levels in both cell lines were not identical, the normalization done for the internalization data by receptor expression (STAR Methods) reduces the impact of receptor expression in the internalization output.

In the absence of GRKs, agonist-induced internalization of ST-mGlu3 was preserved (Figure 4D, red components in the stacked bar graph, not statistically different), and constitutive internalization was even increased when compared with internalization in parental cells (Figure 4D). To assess whether GRKs individually could impact internalization of mGlu3, we overexpressed GRK2 or GRK6, as they are the main GRKs found in HEK293 cells³⁰ and in the human brain.³¹ GRK2 boosted ST-mGlu3 agonist-induced internalization (Figure 4E), while GRK6 boosted both constitutive and agonist-induced ST-mGlu3 internalization (Figure 4F). The GRK effect was kinase dependent, as kinase dead mutants had no effect (Figures S4A-S4C).

As these two GRKs had a different impact on mGlu3 internalization, we investigated the role of all GRKs in the internalization of group I mGlu receptors, of the other group II mGlu2 and mGlu4 as an example of group III receptors. Group I mGlu receptor internalization remains unaltered in the absence of ΔGRKs (Figures S5A and S5B). By deeply examining ST-mGlu5 internalization as representative for group I, only overexpression of GRK2/3 increases its agonist-induced internalization after 60 min at 37°C (Figure 4G) without affecting the constitutive component (Figure 4H). In contrast, ST-mGlu2 kept the GRK preference of group II mGlu receptors for GRK5/6 over
<figure><img src="image_12.png" /><figcaption>**Figure 3.** Only mGlu2-3 among the mGlu3-containing heterodimers internalizes upon agonist stimulation</figcaption></figure>

(A) Schematic of the experimental approach used to measure heterodimer internalization by selectively tagging one of the two subunits of the dimer (black circle, Lumi4-Tb quenched; yellow circle, Lumi4-Tb fluorescence recovery). Green circles represent glutamate.

(B) TR-FRET between the ST-mGlu3 subunit labeled with Lumi4-Tb (T) and HT-mGlu2, HT-mGlu4, HT-mGlu7, or HT-mGlu8 labeled with d2. ΔHTRF(337/665/620; Homogenous Time-Resolved Fluorescence)represents the specific TR-FRET signal calculated by subtracting the TR-FRET signal obtained in non-transfected cells.

(C) Internalization of HT-mGlu2 when co-expressed with mGlu3 in the absence of agonist (basal, 100 µM LY341495) or in the presence of 100 µM glutamate.

(D) Internalization of group III mGlu receptors (HT-mGlu4, HT-mGlu7, or HT-mGlu8) when co-expressed with mGlu3 in the absence of agonist (basal, 100 µM LY341495) or in the presence of 100 µM glutamate.

Data represent the means ± SEM of at least three independent experiments performed in triplicates.

GRK2/3, as significant agonist-induced internalization was revealed in the presence of GRK5/6 but also in the presence of GRK3 (Figure 4G). Overexpression of GRK6 induced agonist-induced internalization of ST-mGlu2 in parental cells, in cells lacking only GRK2 (ΔGRK2), and in ΔGRK cells (Figure S4D), indicating that mGlu2 agonist-induced internalization might be modulated by GRK levels. GRKs have no effect on ST-mGlu4 internalization (Figures 4G and 4H). Taken together, although mGlu receptor internalization can still be detected in the absence of GRKs, GRKs can tune internalization.

### βarrs are required for constitutive internalization

βarr1 and βarr2 bind to GRK-phosphorylated GPCRs to start their internalization (Figure 5A). In accordance, removal of βarr (Arr-2/3-null cell line³² [Δβarr]) blunted agonist-induced internalization of vasopressin receptor 1B (ST-V1b), shown here as a positive control for our assay (Table S1; Figure 5B, red component of the bar) without affecting constitutive internalization (Figure 5B, black component of the bars). Agonist-induced internalization was recovered after overexpression of βarr1 or βarr2 without affecting constitutive internalization (Figure 5B).

As GRKs are not mandatory for mGlu receptor internalization, βarrs were not expected to play a main role in their internalization. Indeed, agonist-induced internalization of ST-mGlu3 was still observed in Δβarr cells (Figure 5C), but constitutive internalization was blunted (Figure 5C) (see Table S1 for mGlu expression in Δβarr cells). Constitutive internalization was recovered by overexpressing βarr2 without affecting agonist-induced internalization (Figure 5D). When looking into the effect of βarrs in group I internalization, we confirmed that βarrs are required for ST-mGlu5 but not for ST-mGlu1 (Figures S5C and S5D, black component of the stacked bars), again without affecting agonist-induced internalization (Figures S5C and S5D, red components of the stacked bars). Still, overexpression of βarr2 could increase agonist-induced internalization of ST-mGlu5 (Figure 5E). Surprisingly, not only constitutive internalization of ST-mGlu3 and ST-mGlu5 was blunted in the absence of βarr, but the constitutive internalization of ST-mGlu2 and ST-mGlu4 was also largely decreased (Figures 5F, S6A, and S6B). βarr2 overexpression rescued constitutive internalization of ST-mGlu2, ST-mGlu3, and ST-mGlu4 but not ST-mGlu5, suggesting that expression of both βarr1 and βarr2 is needed for mGlu5
A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /></figure>

D

<figure><img src="image_16.png" /></figure>

E

<figure><img src="image_17.png" /></figure>

F

<figure><img src="image_18.png" /></figure>

G

<figure><img src="image_19.png" /></figure>

H

<figure><img src="image_20.png" /><figcaption>Figure 4. GRKs fine-tune mGlu receptor internalization</figcaption></figure>

(A) Schematic representing the canonical impact of GRKs in GPCR internalization vs. mGlu receptor internalization. Red circles represent a selective agonist for the studied GPCR.

(B) ST-MOR internalization measured after 60 min at 37°C in the absence of agonist (constitutive [const]) or in the presence of 37.5 µM [D-Ala², N-MePhe³, Gly-ol⁵] enkephalin, DAMGO (agonist-induced [ago]). Experiments were performed in HEK293 cells (control), in HEK293 cells pre-treated with 10 µM cmpd101 for 30 min at 4°C, and in cells lacking of all GRKs under control condition (ΔGRK) or supplemented with a single GRK (+GRK2, +GRK3, +GRK5, and +GRK6). Stacked bars represent constitutive internalization (given by the basal signal, black) and agonist induced (calculated by subtracting the basal signal, red) after 60 min of internalization at 37°C. Data were analyzed using a normality test (Shapiro-Wilk test) subsequent to a one-way ANOVA and followed by Dunnett's post hoc test. ***p ≤ 0.001, **p ≤ 0.01, and ns, *p > 0.05.

(C) Internalization of the ST-mGlu3 receptor in the absence of agonist (basal, 100 µM LY341495) or in the presence of agonist (+Ago, 10 µM LY354740) without or with a pretreatment of 10 µM of cmpd101 for 30 min at 4°C.

(D) Internalization of ST-mGlu3 receptor in the absence (basal, 100 µM LY341495) or in the presence of agonist (10 µM LY354740) in parental or ΔGRK cells.

(E and F) Internalization of ST-mGlu3 in the absence (basal, 100 µM LY341495) or in the presence of agonist (10 µM LY354740) in ΔGRK cells +GRK2 (E) and +GRK6 (F). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test. ***p ≤ 0.001, **p ≤ 0.01, and ns, *p > 0.05. Data represent the means ± SEM of at least three independent experiments performed in triplicates.

(G) Agonist-induced internalization of ST-mGlu5, ST-mGlu2, ST-mGlu3, and ST-mGlu4 after 60 min treatment at 37°C with a selective agonist (10 µM quisqualate for mGlu5, 10 µM LY354740 for mGlu2 and mGlu3, and 10 µM LAP4 for mGlu4) in ΔGRK cells (black) and in ΔGRK cells +GRK2 (blue), +GRK3 (purple), +GRK5 (orange), or +GRK6 (green). Data were analyzed using a normality test (Shapiro-Wilk test) subsequent to a one-way ANOVA and followed by Dunnett's post hoc test. ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns, *p > 0.05.

(H) Constitutive internalization of ST-mGlu5, ST-mGlu2, ST-mGlu3, and ST-mGlu4 after 60 min treatment with 100 µM LY341495 in ΔGRK cells (black) and in ΔGRK cells +GRK2 (blue), +GRK3 (purple), +GRK5 (orange), or +GRK6 (green). Data were analyzed using a normality test

(Shapiro-Wilk test) subsequent to a one-way ANOVA and followed by Dunnett's post hoc test. ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01, ns, *p > 0.05.

Data represent the means ± SEM of at least three independent experiments performed in triplicates.
A

<figure><img src="image_21.png" /></figure>

B

<figure><img src="image_22.png" /></figure>

C

<figure><img src="image_23.png" /></figure>

D

<figure><img src="image_24.png" /></figure>

E

<figure><img src="image_25.png" /></figure>

F

<figure><img src="image_26.png" /><figcaption>Figure 5. βarrs are required for constitutive mGlu receptor internalization</figcaption></figure>

(A) Schematic representing the canonical impact of βarrs in GPCR internalization vs. mGlu receptor internalization.

(B) ST-V1b internalization measured after 60 min at 37°C in the absence of agonist (constitutive) or in the presence of 10 µM of SR149515 (agonist-induced [ago\] in HEK293 cells (control), Δβarr cells, and Δβarr cells and Δβarr cells supplemented with + βarr1 (+βarr1) or βarr2 (+βarr2). Stacked bars represent constitutive internalization (const; given by the basal signal, black) and agonist induced (calculated by subtracting basal signal, red) after 60 min of internalization at 37°C. Data were analyzed using a normality test (Shapiro-Wilk test) subsequent to a one-way ANOVA and followed by Dunnett's post hoc test.
**p ≤ 0.01 and ns, p > 0.05.

(C) Internalization kinetics of the ST-mGlu3 receptor in HEK293 cells (control) and in Δβarr cells in the presence of 100 µM LY341495 (basal) or 10 µM LY354740 (+Ago). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
**p ≤ 0.01 and ns, p > 0.05.

(D) Internalization kinetics of ST-mGlu3 receptor in Δβarr cells supplemented with βarr2 in the presence of 100 µM LY341495 (basal) or 10 µM LY354740 (+Ago). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
***p ≤ 0.001 and ns, p > 0.05.

(E) Agonist-induced internalization of ST-mGlu5 (+10 µM quisqualate), ST-mGlu2 (+10 µM LY354740), ST-mGlu3 (+10 µM LY354740), or ST-mGlu4 (+10 µM L-AP4) after 60 min at 37°C in Δβarr cells (black bars) and in Δβarr cells supplemented with βarr2 (pink bars). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
*p ≤ 0.05 and ns, p > 0.05.

(F) Constitutive internalization of ST-mGlu5, ST-mGlu2, ST-mGlu3, and ST-mGlu4, measured after 60 min at 37°C of incubation with 100 µM LY341495

in Δβarr cells (black bars) and in Δβarr cells supplemented with βarr2 (pink bars). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns, p > 0.05.

Data represent the mean ± SEM of at least three independent experiments performed in triplicate.

constitutive internalization. Indeed, βarr1 was also able to rescue constitutive internalization of ST-mGlu3 (Figure S6D), ST-mGlu2, and ST-mGlu4 (Figures S6B, S6C, S6E, and S6F) but failed to recover ST-mGlu5 constitutive internalization (Figures S6A and S6E). However, it boosted ST-mGlu5 and ST-mGlu3 agonist-induced internalization (Figure S6F).

To further investigate the role of βarrs in mGlu receptor internalization, we studied the recruitment of βarrs by the receptors through bioluminescence resonance energy transfer (BRET). On one hand, we tagged the C-terminal domain of the δ-opioid receptor (DOR), used as a prototypical class A GPCR, and mGlu5, mGlu2, mGlu3, and mGlu4 with the fluorescent protein Venus. On the other hand, we used βarr1 or βarr2 tagged with a luciferase (Renilla luciferase, Rluc). After the addition of the luciferase substrate, coelenterazine, if donor (Rluc) and acceptor (Venus) are in close proximity, a signal of BRET will be detected

(Figure 6A) The constructs (DOR-Venus [DOR-V], mGlu5-V, mGlu2-V, mGlu3-V, and mGlu4-V; Table S1) were used to detect the recruitment of βarr1 or βarr2 tagged with Rluc by following increases of BRET signals. Addition of a selective agonist only increased recruitment of βarr2 by DOR-V without having any effect on mGlu-V receptors (Figure 6B). Interestingly, a large basal BRET was measured between mGlu3-V and βarr2-RLuc, similar to that observed with DOR-V after agonist activation (Figures 6B and 6C), an effect affected neither upon mGlu3 activation (Figure 6B) nor by the antagonist LY341495 (Figure S6H), indicating that it is not due to ambient glutamate. This effect was also observed with βarr1 (Figures S6I–S6K).

As absence of BRET may possibly be due to a non-correct orientation of Rluc and Venus, we further examined the βarr interaction with mGlu receptors using a specific βarr2 antibody labeled with Lumi4-Tb (Figure 6D), as TR-FRET is less sensitive
<figure><img src="image_27.png" /><figcaption>**Figure 6.** The C-terminal domain of mGlu3 is involved in mGlu3 constitutive association with βarr</figcaption></figure>

(A) Schematic representing canonical recruitment of βarr and the BRET strategy proposed to evaluate βarr recruitment to GPCRs.

(B) ST-mGlu-Venus recruitment of βarr2-RLuc, measured by BRET before and after addition of a selective agonist (10 µM quisqualate for mGlu5, 10 µM LY354740 for mGlu2 and mGlu3, and 10 µM LAP4 for mGlu4). Data were normalized by the maximal BRET response obtained by the recruitment of βarr2-RLuc by DOR-Venus in response to 10 µM of the agonist snc-162 (100%) and minimal BRET values in the absence of the agonist of DOR (0%).

(C) ΔBRET expressed in milli net BRET units of the basal BRET (before agonist addition) of DOR-V, mGlu5-V, mGlu2-V, mGlu3-V, and mGlu4-V.

(D) Schematic representing canonical recruitment of βarr and the HTRF strategy proposed to evaluate endogenous βarr2 recruitment to GPCRs tagged with Venus. The yellow circle represents Lumi4-Tb.

(E) ΔHTRF expressed the ratio 520 nm over 620 nm signal, of the basal HTRF signal of DOR-V, mGlu5-V, mGlu2-V, mGlu3-V and mGlu4-V, with 0.5 nM of anti-βarr2-Lumi4-Tb. Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test. \*\*\*p < 0.001 and ns, p > 0.05.

(F) Schematic representing 2C3 and 3C2 chimera constructions. ΔHTRF expressed the ratio 520 nm over 620 nm signal, of the basal HTRF signal of DOR-V, 3C2-V AND 2C3-V, with 0.5 nM of anti-βarr2-Lumi4-Tb.

Data represent the mean ± SEM of at least three independent experiments performed in triplicate.

to orientation constraints. Using this assay, performed after cell permeabilization, a TR-FRET signal could be detected in response to the addition of a DOR agonist (Figure S6L) but also for an agonist-induced recruitment of mGlu3 (Figure S6L). Still, a basal interaction of βarr2 could be detected for mGlu3-V as well as for mGlu2-V, though at a lower level for mGlu2-V, but not with mGlu5-V or mGlu4-V (Figure 6E).

To dissect the molecular determinants underlying this differential interaction of βarrs with mGlu2 and mGlu3 and its potential role in receptor internalization, we engineered chimeric receptors. As GPCRs interact with βarr through the C-terminal domain,³³ we swapped the C-terminal domain of mGlu2 and mGlu3, leading to the chimeras 2C3 (mGlu2 with the C-terminal domain of mGlu3) and 3C2 (mGlu3 with the C-terminal domain of mGlu2; Table S3). As expected, ST-2C3 keeps the internalization properties of ST-mGlu3 either in control and Δβarr cells

(Figure S2B) and is constitutively interacting with βarr (Figure 6F). Also, ST-3C2 internalizes with the profile of ST-mGlu2 (Figure S2C) and shows a much lower basal interaction with βarrs (Figure 6F). Altogether, βarrs are required for constitutive internalization of most mGlu receptors despite the absence of detectable interaction, except for mGlu2 and mGlu3, which constitutively interact with βarr.

## AP2 drives agonist-induced internalization

Finally, we evaluated the role of the tetrameric AP2 protein (α, β, σ, and μ subunits) in mGlu receptor internalization, as AP2 is the final canonical adaptor that binds to βarr and drives endocytosis of agonist-activated GPCRs (Figure 7A). We used small interfering RNA (siRNA) against the μ AP2 subunit that downregulates AP2 expression (Figure S7A) in comparison with irrelevant siRNA (control). siRNA treatment did not impair expression of
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_31.png" /></figure>

treatment with 100 µM LY341495 at 37°C in HEK293 cells (black) and in HEK293 cells treated with siRNA-AP2 (gray). Noteworthy, internalization random fluorescence units (RFUs) were lower than in previous experiments, as DERET was measured 72 h after transfection to find the best compromise of siRNA effect and receptor expression. Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test. *p ≤ 0.05 and ns, p > 0.05. Data represent the mean ± SEM of at least three independent experiments performed in triplicate.

ST-GPCRs (Table S1). As previously described, this down-regulation largely decreased glucagon-like peptide 1 (ST-GLP1) receptor agonist-induced internalization (Figure S7B), as described elsewhere.¹⁸ ST-mGlu3 agonist-induced internalization was almost blunted when downregulating AP2 (Figure 7B) as well as ST-mGlu5 (Figures 7B and S7C). However, we did not observe any significant effect of AP2 downregulation on constitutive internalization of any of the mGlu receptors studied (Figures 7D, S7D, and S7E). AP2 emerges as a crucial adaptor for agonist-induced internalization.

DISCUSSION

Regulating the cell surface density of GPCRs is essential for precisely controlling their physiological function. Extensive studies on rhodopsin-like GPCRs have revealed that agonist-induced receptor internalization involves the sequential action of GRKs, βarrs, and AP2, which drives the activated receptor into clathrin-coated pits. However, alternative mechanisms are also possible, and the internalization that happens in the absence of agonist (constitutive internalization) is an understudied process. Surprisingly, little is known about class C receptors internalization. Our data show that all mGlu receptors undergo constitutive internalization and that only mGlu1, mGlu5 (and likely mGlu1-5), mGlu3, and mGlu2-3 are subject to agonist-induced internalization in HEK293 cells. We found that this process follows a non-canonical mechanism independent of

GRKs and βarrs. Surprisingly, constitutive internalization strictly depends on βarrs except for mGlu1.

Despite years of research on mGlu receptors, the precise mechanism of mGlu receptor internalization remains unclear. Some mGlu receptors have been identified as internalizing in neurons: mGlu1 in Purkinje cells,³⁴ mGlu5 in enteric³⁵ and hippocampal neurons,¹² and mGlu7 and mGlu3 in hippocampal neurons.³⁶,³⁷ These findings highlight internalization as a physiological process that regulates mGlu receptor density at the plasma membrane and, consequently, their response to drugs.

However, the techniques commonly used to detect mGlu receptor internalization present challenges. Antibody-based approaches in living cells can trigger internalization, as demonstrated for MAB1/28 for mGlu7,³⁸ potentially biasing internalization measurements. Additionally, internalization is often analyzed as an endpoint measurement, which may obscure underlying processes such as receptor recycling. Indeed, measuring membrane receptor levels alone may fail to detect internalization if it is counterbalanced by receptor insertion into the plasma membrane or receptor recycling from endosomes.⁹,¹¹ This explains why constitutive internalization of mGlu receptors was not detected in a previous study that systematically analyzed internalization of fluorescently labeled mGlu receptors.³ Moreover, some internalized receptors may remain just beneath the plasma membrane, as seen with the dopamine 1 receptor,³⁹,⁴⁰ making them difficult to detect by microscopy, considering the 200 nm resolution limit. This may explain why mGlu1 and mGlu5 agonist-induced internalization

<caption>**Figure 7. AP2 is required for agonist-induced internalization of mGlu receptors**</caption>

(A) Schematic representing the canonical impact of AP2 in internalization vs. mGlu receptor internalization. The red circle represents the agonist.
(B) Internalization of the ST-mGlu3 receptor in the absence of agonist (control, 100 µM LY341495) or in the presence of agonist (+Ago, 10 µM LY354740) in HEK293 cells treated with irrelevant siRNA (control) vs. HEK293 cells treated with siRNA-AP2. Stacked bars represent constitutive internalization (const; given by the basal signal, black) and agonist induced (ago; calculated by subtracting the basal signal, red) after 60 min of internalization at 37°C. Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
**p ≤ 0.01 and ns, p > 0.05.

(C) Agonist-induced internalization of ST-mGlu5, ST-mGlu2, ST-mGlu3, and ST-mGlu4 after 60 min at 37°C of treatment with a selective agonist (10 µM quisqualate for mGlu5, 10 µM LY354740 for mGlu2 and mGlu3, and 10 µM LAP4 for mGlu4) in HEK293 cells treated with irrelevant siRNA (red) and in HEK293 cells treated with siRNA-AP2 (orange). Data were analyzed using a normality test (Shapiro-Wilk test) followed by two-tailed Student's t test.
**p ≤ 0.01 and ns, p > 0.05.

(D) Constitutive internalization of ST-mGlu5, ST-mGlu2, ST-mGlu3, and ST-mGlu4 after 60 min of
could not be detected by simple observation of the fluorescently labeled receptors in a recent study,⁹ even though mGlu1 and mGlu5 are widely accepted as undergoing agonist-induced internalization in HEK293 cells.¹²,⁴¹⁻⁴³ Also, in this study, the authors reported agonist-induced internalization of mGlu7 and mGlu8, in contrast to our finding, but this was mainly observed with overexpressed GRK2.

To overcome these caveats, we optimized internalization measurements by DERET. DERET has been successfully employed to track the internalization of already more than 50 GPCRs,¹⁸ including mGlu5,¹⁷,⁴³ whose internalization has been shown to be allosterically modulated.⁴⁴,⁴⁵ Using DERET, we identified both constitutive and agonist-induced internalization of mGlu receptors over time and demonstrated that their internalization kinetics vary significantly. Specifically, we observed that mGlu5 rapidly recycles to the plasma membrane after agonist-induced internalization, which may explain discrepancies observed in endpoint measurements.⁹

Among all mGlu receptors, only mGlu1, mGlu5, and mGlu3 homodimers undergo agonist-induced internalization in HEK293 cells, whereas mGlu2 and group III receptors do not. Consequently, we expect mGlu1-5 heterodimers to internalize upon agonist stimulation, while group III heterodimers likely do not. Interestingly, the mGlu3 subunit drives mGlu2 internalization upon agonist stimulation when assembled into a mGlu2-3 heterodimer but not when paired with mGlu4, mGlu7, or mGlu8. This suggests that the C-terminal domain of mGlu2 does not restrict receptor surface localization, unlike those of group III mGlu receptors. These findings underscore the functional significance of mGlu receptor heterodimerization⁴⁶ and the importance of considering subunit composition when analyzing internalization.

The interpretation of mGlu receptor internalization is further complicated by variations in cellular models across studies. HEK293 cells express mostly GRK2 and GRK6,³⁰ similar to neurons,³¹ but GRK4 has been described to participate in mGlu1 internalization.¹⁰ Additionally, HEK293 cells lack neuron-specific internalization adaptors such as PICK-1³⁷,⁴⁷ or norbin.⁴⁸ It may explain why we did not observe agonist-induced internalization of mGlu7 despite its reported internalization in neurons.³⁶ Also, the expression level of internalization adaptors such as GRKs may vary from cell to cell and even among neuronal types, as already demonstrated in postmortem human brain tissue.³¹ GRK availability could provide an explanation for any potential difference in the internalization properties of mGlu receptors. For example, a differential expression level of GRK2 in striatal versus cortical neurons may explain the differential internalization properties of mGlu5, agonist-induced internalization being observed mainly in striatal neurons that express more GRK2.⁸

To ensure accurate comparisons, we examined the internalization properties of all mGlu receptors in the same cellular background using a real-time analysis technique. We showed that GRKs are dispensable for constitutive and agonist-induced mGlu receptor internalization. Still, they modulate the process; GRK2 and GRK3 specifically influence mGlu5 internalization kinetics, and mGlu2 undergoes agonist-induced internalization, likely through GRK5/6 phosphorylation, but not in the presence of GRK2, with the latter possibly inhibiting the effect of GRK6. This suggests that mGlu5 and mGlu2 have a GRK preference

that could be masked in HEK293 cells due to the fact that both GRK2 and GRK6 are expressed at the same level.³⁰ This can be explained by different phosphorylation codes generated by these different GRKs. Also, GRK overexpression affects mGlu3 internalization dynamics with a strong potentiation of the constitutive internalization of this receptor in the absence of GRKs. These GRK-specific effects are not surprising, given that GRK2/3 are cytoplasmic, whereas GRK5/6 are membrane-bound.⁴⁹ To be noted, our study was done mainly with the endogenous levels of GRKs expressed in HEK293 cells, except for the GRK study. Overexpression of any GRK in HEK293 cells can alter the internalization profile of the mGlu receptor under study, as shown in Figure S4D for mGlu2. GRK expression levels could explain differences observed with previous studies, where mGlu8 has been detected to undergo agonist-induced internalization in HEK293 cells overexpressing GRK2,¹¹,⁵⁰ in contrast with our observations (Figure 2). Also, the mGlu7 internalization reported in neurons may be due to a higher GRK2 expression than in the HEK293 cells.⁸ Finally, the availability of GRKs may determine the ability to phosphorylate mGlu receptors and induce internalization, as observed with opioid receptors.⁵¹

In contrast to canonical GPCR internalization, agonist-induced internalization of mGlu receptors does not require βarrs, consistent with previous reports.⁵²,⁵³ Although βarr involvement in mGlu1 internalization was suggested in a study using a dominant-negative construct, that study did not separate the constitutive and agonist-induced internalization.¹⁰ Of interest, βarrs are known to bind within the cavity created by the movement of transmembrane domain 6 (TM6) in activated class A GPCRs,⁵⁴ a cavity that cannot exist in class C GPCRs, as no movement of TM6 is observed upon activation.⁵⁵,⁵⁶

Nonetheless, our study shows that βarrs are required for the constitutive internalization of several mGlu receptors in HEK293 cells, as observed in other class C GPCR like the γ-aminobutyric acid B (GABA<sub>B</sub>) receptor. The GABA<sub>B</sub> receptor undergoes constitutive, clathrin-dependent internalization in neurons.⁵⁷ Contrary to this, mGlu7 has been shown in the literature to undergo constitutive internalization in a clathrin-independent manner.⁵⁸ We showed that mGlu5, mGlu2, mGlu3, and mGlu4 rely on βarr to efficiently pursue constitutive internalization, in contrast to mGlu1. Even if mGlu5 and mGlu1 belong to the same group of mGlu receptors, their internalization mechanisms differ, as already illustrated by others, who have shown the unequal role of PICK1 in group I mGlu receptors.⁴⁷

In the case of group II mGlu receptors, mGlu3, and mGlu2, βarr's role in constitutive internalization can be the consequence of the constitutive interaction of βarr with these receptors, similar to V1b,⁵⁹ atypical chemokine receptor 4 (ACKR4),⁶⁰ or C-C chemokine receptor type 1 (CCR1).⁶¹ In the case of mGlu5 and group III mGlu receptors, for which no interaction with βarr could be detected, one can propose at least two explanations. One is that the βarr interaction may be transient on these receptors, making it impossible to detect by a BRET or TR-FRET approach, considering there is no way to synchronize this effect on every receptor at a given time when analyzing the constitutive internalization. The other one is that βarrs may participate indirectly in the internalization process, through other protein partners, and possibly also transiently. This could prevent any detection with our assays.
Given that βarrs are not involved in mGlu receptor agonist-induced internalization, AP2 should theoretically play no role according to canonical internalization, as AP2 is brought to the GPCR complex through its interaction with βarrs. Intriguingly, we found that AP2 is not required for constitutive mGlu receptor internalization despite the latter being, in most cases, βarr dependent. However, AP2 is essential for agonist-induced internalization of mGlu5 and mGlu3. This suggests that mGlu5 and mGlu3 receptors may directly recruit AP2, independent of βarrs, as observed with GLP1.¹⁸

In this work, we precisely identified the constitutive and the agonist-induced mechanisms of mGlu receptors in HEK293 cells. We demonstrated that constitutive internalization relies on βarrs, whereas mGlu5 and mGlu3 agonist-induced internalization is dependent on AP2. GRKs serve as fine-tuning modulators of the internalization kinetics rather than as indispensable components. Nevertheless, mGlu receptors could follow diverse internalization mechanisms in neurons, as already suggested elsewhere for mGlu5.⁶² This study provides a solid foundation for evaluating mGlu receptor internalization across different cell types, such as brain cells.

## Limitations of the study

Taken together, these results revealed that both constitutive and agonist-induced internalization of mGlu receptors involve mechanisms different from those used by many class A GPCRs. Although some of the partners involved in the canonical internalization are also involved for mGlu receptors, their roles clearly differ. Indeed, GRKs modulate the mGlu receptor process, while they are required for many class A GPCRs. How is this possible? Which additional partners are needed for these regulatory effects? Many questions are raised that need to be clarified. It is important to highlight that HEK293 cells do not express GRK4²⁹; its role in mGlu receptor internalization has not been assessed in this study. Future work will be needed to ascertain the role of GRK4 in mGlu receptor internalization, as its role in mGlu desensitization has been reported.¹⁰ The observation that βarrs are needed for the constitutive internalization and not for the agonist-induced one was not expected; and additional work is needed to solve this process at the molecular level. The role of the AP2 complex, independent of βarrs, suggests a direct interaction with the mGluRs, as observed with GLP1R,¹⁸ or via an unidentified partner. More work is then needed to clarify how AP2 controls mGlu internalization. Of note, our data revealed different scenarios for all five mGlu receptors studied (mGlu1, mGlu5, mGlu2, mGlu3, and mGlu4), suggesting that the other mGlu receptors may also reveal additional surprises. Although our work revealed that mGlu receptor internalization processes are peculiar and differ from those of many others GPCRs, the real mechanism involved remains to be elucidated. One will be tempted to study the role of caveolae, identified for mGlu5 internalization in neurons,¹² but other partners not yet identified will have to be discovered.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Jean-Philippe Pin (jean-philippe.pin@igf.cnrs.fr).

## Materials availability

This study did not generate new unique reagents.

## Data and code availability

* Data and linked metadata used to create all figures have been deposited in Zenodo (https://doi.org/10.5281/zenodo.15479046).

* This study does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank all members of Rondard's team for fruitful discussions. We thank the Arpège platform of the Institute of Functional Genomics for providing facilities and technical support and Revvity for providing reagents. M.C. was supported by a postdoctoral fellowship granted by the Alfonso Martin Escudero Foundation (FAME) and a Marie Sklodowska-Curie Individual Fellowship from the European Union's Horizon 2020 Research and Innovation Program to project GLUGLU (project 101065171). M.H. was the recipient of a doctoral fellowship from the University of Montpellier (2024). P.-A.L. was the recipient of a post-doctoral fellowship from the LabEx Mablprove (grant NR-10-LABX-5301). This work was supported by Fondation pour la Recherche Médicale (DEQ20170336747 to J.-P.P. and EQU202303016470 to P.R.), the Agence Nationale de la Recherche (ANR-22-CE16-0022-01 to J.-P.P.), and Revvity (Codolet, France).

## AUTHOR CONTRIBUTIONS

M.C. conceptualized the project, performed experiments and data analysis, and wrote the manuscript. J.L. performed the western blot. D.M. and I.B. set the basis with preliminary data. J.D., C.H., and A.I. provided the cell lines. M.H. provided experimental support. P.R. got funding for the project. P.-A.L. gave a critical review and constructive discussions. J.-P.P. and L.P. conceptualized the project, supervised the work, and wrote the manuscript. All authors approved the manuscript.

## DECLARATION OF INTERESTS

P.R. and J.-P.P. are funded by Revvity through the collaborative laboratory Eidos.

## DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

During the revision of this work, the authors used ChatGPT to minimize grammar mistakes. In any case, the text was reviewed afterward, and the authors take full responsibility for the content of the publication.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* METHOD DETAILS

* Silencing of protein expression

* DERET (internalization assay)

* Recycling measurements (DERET)

* TR-FRET for heterodimer detection

* BRET for βarr recruitment detection

* HTRF for endogenous β-arrestin2 recruitment

* Western blot

* QUANTIFICATION AND STATISTICAL ANALYSIS

* Data analysis

* Statistical analysis
## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116068.

Received: January 17, 2025

Revised: June 23, 2025

Accepted: July 8, 2025

## REFERENCES

1. Hauser, A.S., Attwood, M.M., Rask-Andersen, M., Schiöth, H.B., and Gliori, D.E. (2017). Trends in GPCR drug discovery: new agents, targets, indications. Nat. Rev. Drug Discov. 16, 829-842. https://doi.org/10.1038/nrd.2017.178.

2. Kaksonen, M., and Roux, A. (2018). Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313-326. https://doi.org/10.1038/nrm.2017.132.

3. Moo, E.V., van Senten, J.R., Bräuner-Osborne, H., and Møller, T.C. (2021). Arrestin-Dependent and -Independent Internalization of G Protein-Coupled Receptors: Methods, Mechanisms, and Implications on Cell Signaling. Mol. Pharmacol. 99, 242-255. https://doi.org/10.1124/MOLPHARM.120.000192.

4. Tian, X., Kang, D.S., and Benovic, J.L. (2014). β-arrestins and G Protein-Coupled Receptor Trafficking. Handb. Exp. Pharmacol. 219, 173-186. https://doi.org/10.1007/978-3-642-41199-1_9.

5. Scarselli, M., and Donaldson, J.G. (2009). Constitutive internalization of G protein-coupled receptors and G proteins via clathrin-independent endocytosis. J. Biol. Chem. 284, 3577-3585. https://doi.org/10.1074/JBC.M806819200.

6. Spiess, K., Bagger, S.O., Torz, L.J., Jensen, K.H.R., Walser, A.L., Kvam, J. M., Møgelmose, A.S.K., Daugvilaite, V., Junnila, R.K., Hjorto, G.M., and Rosenkilde, M.M. (2019). Arrestin-independent constitutive endocytosis of GPR125/ADGRA3. Ann. N. Y. Acad. Sci. 1456, 186-199. https://doi.org/10.1111/NYAS.14263.

7. Iacovelli, L., Nicoletti, F., and De Blasi, A. (2013). Molecular mechanisms that desensitize metabotropic glutamate signaling: an overview. Neuropharmacology 66, 24-30. https://doi.org/10.1016/J.NEUROPHARM.2012.05.005.

8. Ribeiro, F.M., Ferreira, L.T., Paquet, M., Cregan, T., Ding, Q., Gros, R., Ferguson, S.S.G., and Taylor, A. (2009). Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 5 Desensitization and Internalization by G Protein-coupled Receptor Kinase 2 in Neurons * From the. J. Biol. Chem. 284, 23444-23453. https://doi.org/10.1074/jbc.M109.000778.

9. Abreu, N., Acosta-Ruiz, A., Xiang, G., and Levitz, J. (2021). Mechanisms of differential desensitization of metabotropic glutamate receptors. Cell Rep. 35, 109050. https://doi.org/10.1016/J.CELREP.2021.109050.

10. Mundell, S.J., Pula, G., McIlhinney, R.A.J., Roberts, P.J., and Kelly, E. (2004). Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors. Biochemistry 43, 7541-7551. https://doi.org/10.1021/BI0359022.

11. Lee, J., Gonzalez-Hernandez, A.J., Kristt, M., Abreu, N., Roßmann, K., Arefin, A., Marx, D.C., Broichhagen, J., and Levitz, J. (2023). Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers. Sci. Adv. 9, eadi8076. https://doi.org/10.1126/SCIADV.ADI8076.

12. Fourgeaud, L., Bessis, A.S., Rossignol, F., Pin, J.P., Olivo-Marin, J.C., and Hémar, A. (2003). The metabotropic glutamate receptor mGluR5 is endocytosed by a clathrin-independent pathway. J. Biol. Chem. 278, 12222-12230. https://doi.org/10.1074/jbc.M205663200.

13. Møller, T.C., Moo, E.V., Inoue, A., Pedersen, M.F., and Bräuner-Osborne, H. (2024). Characterization of the real-time internalization of nine GPCRs

reveals distinct dependence on arrestins and G proteins. Biochim. Biophys. acta. Mol. cell Res 1871, 119584. https://doi.org/10.1016/J.BBAMCR.2023.119584.

14. Dhami, G.K., Anborgh, P.H., Dale, L.B., Sterne-Marr, R., and Ferguson, S.S. G. (2002). Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor Signaling by G Protein-coupled Receptor Kinase 2. J. Biol. Chem. 277, 25266-25272. https://doi.org/10.1074/jbc.M203593200.

15. Crupi, R., Impellizzeri, D., and Cuzzocrea, S. (2019). Role of Metabotropic Glutamate Receptors in Neurological Disorders. Front. Mol. Neurosci. 12, 20. https://doi.org/10.3389/FNMOL.2019.00020.

16. Adams, D.H., Zhang, L., Millen, B.A., Kinon, B.J., and Gomez, J.-C. (2014). Pomaglumetad Methionil (LY2140023 Monohydrate) and Aripiprazole in Patients with Schizophrenia: A Phase 3, Multicenter, Double-Blind Comparison. Schizophr. Res. Treatment 201, 758212. https://doi.org/10.1155/2014/758212.

17. Levoye, A., Zwier, J.M., Jaracz-Ros, A., Klipfel, L., Cottet, M., Maurel, D., Bdioui, S., Balabanian, K., Prézeau, L., Trinquet, E., et al. (2015). A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate. Front. Endocrinol. 6, 167. https://doi.org/10.3389/fendo.2015.00167.

18. Liu, J., Xue, L., Ravier, M., Inoue, A., Drube, J., Hoffmann, C., Trinquet, E., Dupuis, E., Prézeau, L., Pin, J.-P., and Rondard, P. (2024). Multi-faceted roles of β-arrestins in G protein-coupled receptors endocytosis. Preprint at bioRxiv. https://doi.org/10.1101/2024.01.18.576020.

19. Hoare, S.R.J., Tewson, P.H., Quinn, A.M., Hughes, T.E., and Bridge, L.J. (2020). Analyzing kinetic signaling data for G-protein-coupled receptors. 23. Sci. Rep. 10, 12263. https://doi.org/10.1038/s41598-020-67844-3.

20. Acher, F. (2011). Metabotropic glutamate receptors. Tocris Biosci. Rev. Lett. 26, 1-10.

21. Tora, A.S., Rovira, X., Cao, A.M., Cabayé, A., Olofsson, L., Malhaire, F., Scholler, P., Baik, H., Van Eeckhaut, A., Smolders, I., et al. (2018). Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3. Neuropharmacology 140, 275-286. https://doi.org/10.1016/j.neuropharm.2018.08.011.

22. Doumazane, E., Scholler, P., Zwier, J.M., Trinquet, E., Rondard, P., and Pin, J.P. (2011). A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors. FASEB J. 25, 66-77. https://doi.org/10.1096/fj.10-163147.

23. Kniazeff, J., Bessis, A.S., Maurel, D., Ansanay, H., Prézeau, L., and Pin, J. P. (2004). Closed state of both binding domains of homodimeric mGlu receptors is required for full activity. Nat. Struct. Mol. Biol. 11, 706-713. https://doi.org/10.1038/nsmb794.

24. von Zastrow, M., and Williams, J.T. (2012). Modulating neuromodulation by receptor membrane traffic in the endocytic pathway. Neuron 76, 22-32. https://doi.org/10.1016/J.NEURON.2012.09.022.

25. Perkovska, S., Méjean, C., Ayoub, M.A., Li, J., Hemery, F., Corbani, M., Laguette, N., Ventura, M.A., Orcel, H., Durroux, T., et al. (2018). V1b vasopressin receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase. Traffic 19, 58-82. https://doi.org/10.1111/TRA.12535.

26. Meng, J., Xu, C., Lafon, P.A., Roux, S., Mathieu, M., Zhou, R., Scholler, P., Blanc, E., Becker, J.A.J., Le Merrer, J., et al. (2022). Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain. Nat. Chem. Biol. 18, 894-903. https://doi.org/10.1038/S41589-022-01050-2.

27. Ferraguti, F., and Shigemoto, R. (2006). Metabotropic glutamate receptors. Cell Tissue Res. 326, 483-504. https://doi.org/10.1007/s00441-006-0266-5.

28. Møller, T.C., Pedersen, M.F., van Senten, J.R., Seiersen, S.D., Mathiesen, J.M., Bouvier, M., and Bräuner-Osborne, H. (2020). Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells. Sci. Rep. 10, 17395. https://doi.org/10.1038/S41598-020-73674-0.
29. Drube, J., Haider, R.S., Matthees, E.S.F., Reichel, M., Zeiner, J., Fritz-wanker, S., Ziegler, C., Barz, S., Klement, L., Filor, J., et al. (2022). GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation. Nat. Commun. **13**, 540. https://doi.org/10.1038/S41467-022-28152-8.

30. Reichel, M., Weitzel, V., Klement, L., Hoffmann, C., and Drube, J. (2022). Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots. Int. J. Mol. Sci. **23**, 1195. https://doi.org/10.3390/IJMS23031195.

31. Guimarães, T.R., Swanson, E., Kofler, J., and Thathiah, A. (2021). G protein-coupled receptor kinases are associated with Alzheimer's disease pathology. Neuropathol. Appl. Neurobiol. **47**, 942-957. https://doi.org/10.1111/NAN.12742.

32. Alvarez-Curto, E., Inoue, A., Jenkins, L., Raihan, S.Z., Prihandoko, R., Tobin, A.B., and Milligan, G. (2016). Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling. J. Biol. Chem. **291**, 27147-27159. https://doi.org/10.1074/JBC.M116.754887.

33. Zhou, X.E., He, Y., de Waal, P.W., Gao, X., Kang, Y., Van Eps, N., Yin, Y., Pal, K., Goswami, D., White, T.A., et al. (2017). Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors. Cell **170**, 457-469.e13. https://doi.org/10.1016/j.cell.2017.07.002.

34. Sallese, M., Salvatore, L., D'Urbano, E., Sala, G., Storto, M., Launey, T., Nicoletti, F., Knöpfel, T., and De Blasi, A. (2000). The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1. FASEB J. **14**, 2569-2580. https://doi.org/10.1096/FJ.00-0072COM.

35. Liu, M., and Kirchgessner, A.L. (2000). Agonist- and Reflex-Evoked Internalization of Metabotropic Glutamate Receptor 5 in Enteric Neurons. J. Neurosci. **20**, 3200-3205. https://doi.org/10.1523/JNEUROSCI.20-09-03200.2000.

36. Pelkey, K.A., Yuan, X., Lavezzari, G., Roche, K.W., and McBain, C.J. (2007). mGluR7 undergoes rapid internalization in response to activation by the allosteric agonist AMN082. Neuropharmacology **52**, 108-117. https://doi.org/10.1016/j.neuropharm.2006.07.020.

37. Tuduri, P., Bouquier, N., Girard, B., Moutin, E., Thouaye, M., Perroy, J., Bertaso, F., and Ster, J. (2022). Modulation of Hippocampal Network Oscillation by PICK1-Dependent Cell Surface Expression of mGlu3 Receptors. J. Neurosci. **42**, 8897-8911. https://doi.org/10.1523/JNEUROSCI.0063-22.2022.

38. Ullmer, C., Zoffmann, S., Bohrmann, B., Matile, H., Lindemann, L., Flor, P. J., and Malherbe, P. (2012). Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7. Br. J. Pharmacol. **167**, 1448-1466. https://doi.org/10.1111/j.1476-5381.2012.02090.x.

39. Kotowski, S.J., Hopf, F.W., Seif, T., Bonci, A., and von Zastrow, M. (2011). Endocytosis Promotes Rapid Dopaminergic Signaling. Neuron **71**, 278-290. https://doi.org/10.1016/j.neuron.2011.05.036/ATTACHMENT/D038C525-3485-4245-9904-BC0414BE444F/MMC4.MOV.

40. Gómez-Santacana, X., Boutonnet, M., Martínez-Juvés, C., Cimadevila, M., Catena, J., Moutin, E., Roux, T., Trinquet, E., Lamarque, L., Perroy, J., et al. (2025). A modular click ligand-directed approach to label endogenous dopamine D1 receptors in live cells. Commun. Chem. **8**, 113. https://doi.org/10.1038/S42004-025-01504-3.

41. Francesconi, A., Kumari, R., and Zukin, R.S. (2009). Regulation of Group I Metabotropic Glutamate Receptor Trafficking and Signaling by the Caveolar/Lipid Raft Pathway. J. Neurosci. **29**, 3590-3602. https://doi.org/10.1523/JNEUROSCI.5824-08.2009.

42. Gulia, R., Sharma, R., and Bhattacharyya, S. (2017). A critical role for ubiquitination in the endocytosis of glutamate receptors. J. Biol. Chem. **292**, 1426-1437. https://doi.org/10.1074/JBC.M116.752105/ASSET/2305FED5-63EC-4F80-BFF0-F79F2A9CDA7F/MAIN.ASSETS/GR7.JPG.

43. van Senten, J.R., Møller, T.C., Moo, E.V., Seiersen, S.D., and Bräuner-Osborne, H. (2022). Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. J. Biol. Chem. **298**, 102466. https://doi.org/10.1016/j.jbc.2022.102466.

44. Arsova, A., Møller, T.C., Vedel, L., Hansen, J.L., Foster, S.R., Gregory, K. J., and Bräuner-Osborne, H. (2020). Detailed in Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5. Mol. Pharmacol. **98**, 49-60. https://doi.org/10.1124/mol.119.119032.

45. Arsova, A., Møller, T.C., Hellyer, S.D., Vedel, L., Foster, S.R., Hansen, J.L., Bräuner-Osborne, H., and Gregory, K.J. (2021). Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias. Mol. Pharmacol. **99**, 328-341. https://doi.org/10.1124/MOLPHARM.120.000185.

46. Belkacemi, K., Rondard, P., Pin, J.P., and Prézeau, L. (2025). Heterodimers Revolutionize the Field of Metabotropic Glutamate Receptors. Neuroscience **578**, 46-58. https://doi.org/10.1016/J.NEUROSCIENCE.2024.06.013.

47. Ramsakha, N., Ojha, P., Pal, S., Routh, S., Citri, A., and Bhattacharyya, S. (2023). A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis. J. Biol. Chem. **299**, 104837. https://doi.org/10.1016/J.JBC.2023.104837.

48. Ojha, P., Pal, S., and Bhattacharyya, S. (2022). Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin. J. Neurosci. **42**, 731-748. https://doi.org/10.1523/JNEUROSCI.1037-21.2021.

49. Ribas, C., Penela, P., Murga, C., Salcedo, A., García-Hoz, C., Jurado-Pueyo, M., Aymerich, I., and Mayor, F. (2007). The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim. Biophys. Acta **1768**, 913-922. https://doi.org/10.1016/j.BBAMEM.2006.09.019.

50. Marx, D.C., Gonzalez-Hernandez, A.J., Huynh, K., Strauss, A., Rico, C., Siepe, D., Gallo, P., Lee, J., Sharghi, S., Arefin, A., et al. (2025). Structural Diversity of Metabotropic Glutamate Receptor/Beta-Arrestin Coupling. Preprint at bioRxiv. https://doi.org/10.1101/2025.02.03.636340.

51. Jullié, D., Benitez, C., Knight, T.A., Simic, M.S., and von Zastrow, M. (2022). Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling. eLife **11**, e81298. https://doi.org/10.7554/ELIFE.81298.

52. Dhami, G.K., and Ferguson, S.S.G. (2006). Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis. Pharmacol. Ther. **111**, 260-271. https://doi.org/10.1016/j.PHARMOTHERA.2005.01.008.

53. Pin, J.P., and Bettler, B. (2016). Organization and functions of mGlu and GABA B receptor complexes. Nature **540**, 60-68. https://doi.org/10.1038/nature20566.

54. Rasmussen, S.G.F., Devree, B.T., Zou, Y., Kruse, A.C., Chung, K.Y., Kolbikka, T.S., Thian, F.S., Chae, P.S., Pardon, E., Calinski, D., et al. (2011). Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature **477**, 549-555. https://doi.org/10.1038/nature10361.

55. Shen, C., Mao, C., Xu, C., Jin, N., Zhang, H., Shen, D.D., Shen, Q., Wang, X., Hou, T., Chen, Z., et al. (2021). Structural basis of GABAB receptor-Gi protein coupling. Nat **594**, 594-598. https://doi.org/10.1038/s41586-021-03507-1.

56. Seven, A.B., Barros-Álvarez, X., Lapeyrière, M. de, Papasergi-Scott, M. M., Robertson, M.J., Zhang, C., Nwokonko, R.M., Gao, Y., Meyerowitz, J.G., Rocher, J.-P., et al. (2021). G-protein activation by a metabotropic glutamate receptor. Nature **2021**, 1-5. https://doi.org/10.1038/s41586-021-03680-3.

57. Vargas, K.J., Terunuma, M., Tello, J.A., Pangalos, M.N., Moss, S.J., and Couve, A. (2008). The Availability of Surface GABA B Receptors Is Independent of Aminobutyric Acid but Controlled by Glutamate in Central
Neurons * □ S. J. Biol. Chem. 283, 24641-24648. https://doi.org/10.1074/jbc.M802419200.

58. Lavezzari, G., and Roche, K.W. (2007). Constitutive endocytosis of the metabotropic glutamate receptor mGluR7 is clathrin-independent. *Neuropharmacology* **52**, 100–107. https://doi.org/10.1016/j.neuropharm.2006.07.011.

59. Koshimizu, T.A., Honda, K., Nagaoka-Uozumi, S., Ichimura, A., Kimura, I., Nakaya, M., Sakai, N., Shibata, K., Ushijima, K., Fujimura, A., et al. (2018). Complex formation between the vasopressin 1b receptor, β-arrestin-2, and the μ-opioid receptor underlies morphine tolerance. *Nat. Neurosci.* **21**, 820–833. https://doi.org/10.1038/s41593-018-0144-y.

60. Matti, C., Salnikov, A., Artinger, M., D'Agostino, G., Kindinger, I., Uguccioni, M., Thelen, M., and Legler, D.F. (2020). ACKR4 Recruits

GRK3 Prior to β-Arrestins but Can Scavenge Chemokines in the Absence of β-Arrestins. Front. Immunol. 11, 720. https://doi.org/10.3389/fimmu.2020.00720.

61. Gilliland, C.T., Salanga, C.L., Kawamura, T., Trejo, J., and Handel, T. M. (2013). The chemokine receptor CCR1 is constitutively active, which leads to G protein-independent, β-arrestin-mediated internalization. *J. Biol. Chem.* **288**, 32194-32210. https://doi.org/10.1074/JBC.M113.503797.

62. Scheefhals, N., Catsburg, L.A.E., Westerveld, M.L., Blanpied, T.A., Hoo-genraad, C.C., and MacGillavry, H.D. (2019). Shank Proteins Couple the Endocytic Zone to the Postsynaptic Density to Control Trafficking and Signaling of Metabotropic Glutamate Receptor 5. Cell Rep. 29, 258-269. e8. https://doi.org/10.1016/j CELREP.2019.08.102.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>mouse anti-AP2 m2</td><td>BD Biosciences</td><td>RRID:AB_398873</td></tr><tr><td>mouse anti-b actin</td><td>Sigma Aldrich</td><td>RRID:AB_476744</td></tr><tr><td>rabbit HTRF AbAnti-Mouse IgG d2-Conjugate</td><td>Revvity</td><td>RRID: AB_3711517</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>LY341495 disodium salt</td><td>Tocris Bioscience</td><td>#4062</td></tr><tr><td>L-Quisqualic acid</td><td>Tocris Bioscience</td><td>#0188</td></tr><tr><td>LY354740</td><td>Tocris Bioscience</td><td>#3246</td></tr><tr><td>DAMGO</td><td>Tocris Bioscience</td><td>#1171</td></tr><tr><td>Exendin-4</td><td>Tocris Bioscience</td><td>#1933</td></tr><tr><td>L-AP4</td><td>Tocris Bioscience</td><td>#0130</td></tr><tr><td>DMEM</td><td>Gibco (Thermo Fisher Scientific)</td><td>#41965-039</td></tr><tr><td>DMEM + GlutaMAX™-I</td><td>Gibco (Thermo Fisher Scientific)</td><td>#31966-021</td></tr><tr><td>Opti-MEM®</td><td>Gibco (Thermo Fisher Scientific)</td><td>#31985-047</td></tr><tr><td>Trypsine-EDTA (0.05%)</td><td>Gibco (Thermo Fisher Scientific)</td><td>#25300-054</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>#11668019</td></tr><tr><td>Coelenterazine-h</td><td>Promega Corporation</td><td>#S2011</td></tr><tr><td>DPBS</td><td>Sigma Aldrich</td><td>#D8537</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>#F7524</td></tr><tr><td>L-glutamic acid</td><td>Sigma Aldrich</td><td>#G8415</td></tr><tr><td>Fluorescein sodium</td><td>Sigma Aldrich</td><td>#46955</td></tr><tr><td>Cmpd101 hydrochloride</td><td>Sigma Aldrich</td><td>#SML2784</td></tr><tr><td>96 well plate, white</td><td>Greiner</td><td>#655083</td></tr><tr><td>DharmaFECT1</td><td>Horizon Discovery</td><td>#T-2001-04</td></tr><tr><td>Tag-lite SNAP-Lumi4-Tb Labeling Reagent</td><td>Revvity</td><td>#SSNPTBD</td></tr><tr><td>Tag-lite HaloTag-Lumi4-Tb Labeling Reagent</td><td>Revvity</td><td>#SHALOTBC</td></tr><tr><td>Tag-lite CLIP-Red Labeling Reagent</td><td>Revvity</td><td>#SCLPREDF</td></tr><tr><td>Tag-lite HaloTag-Red Labeling Reagent</td><td>Revvity</td><td>#SHALOREDE</td></tr><tr><td>5xTag-lite SNAP/CLIP Labeling Medium</td><td>Revvity</td><td>#LABMED</td></tr><tr><td>RIPA Buffer</td><td>Thermo Fisher</td><td>#89900</td></tr><tr><td>Protease Inhibitor cocktail</td><td>Merck</td><td>#5892953001</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Beta-Arrestin recruitment kit</td><td>Revvity</td><td>#62BDBAR2PEB</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>Source data for all figures and supplementary figures</td><td>This paper</td><td>https://doi.org/10.5281/zenodo.15479046</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>HEK293</td><td>ATCC</td><td>#CRL-1573TM</td></tr><tr><td>ΔQ-GRK cells</td><td>Gift from Dr. C Hoffmann</td><td>N/A</td></tr><tr><td>ΔGRK2 cells</td><td>Gift from Dr. C Hoffmann</td><td>N/A</td></tr><tr><td>ΔβArr1/2 HEK293</td><td>Gift from Dr. A Inoue</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>control-siRNA</td><td>Horizon Discovery</td><td>#D-001810-10-20</td></tr><tr><td>ON-TARGETplus Human AP2-μ2subunit</td><td>Horizon Discovery</td><td>#L-008170-00-0005</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu1 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu2 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu3 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu4 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu5 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu5-YADA (rat)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-CLIP-mGlu5 (rat)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu6 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu7 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu8 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-GLP1 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-MOR (human)</td><td>Liu et al.<sup>18</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-V1b (human)</td><td>Perkovska et al.<sup>25</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-Halo-mGlu2 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-Halo-mGlu4 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-Halo-mGlu7 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-Halo-mGlu8 (human)</td><td>Doumazane et al.<sup>22</sup></td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu2(human)-Venus</td><td>GeneCust</td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu3(human)-Venus</td><td>GeneCust</td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu4(human)-Venus</td><td>GeneCust</td><td>N/A</td></tr><tr><td>pcDNA3-myc-mGlu5(human)-Venus</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3-DOR(human)-Venus</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA-β-arrestin1(human)-RLuc8</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA-β-arrestin2(human)-RLuc8</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK2(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK3(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK5(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK6(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu2C3</td><td>GeneCust</td><td>N/A</td></tr><tr><td>pcDNA3.1-FLAG-SNAP-mGlu3C2</td><td>GeneCust</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK2K220R(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pcDNA3.1-GRK6K215R(human)</td><td>ARPEGE platform, France</td><td>N/A</td></tr><tr><td>pRK-EAAC1</td><td>Tora et al.<sup>21</sup></td><td>N/A</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>GraphPad Prism software version 10.0.0 for Windows</td><td>GraphPad Software</td><td>www.graphpad.com</td></tr><tr><td>Pharmechanics equations</td><td>Hoare et al.<sup>19</sup></td><td>https://drive.google.com/drive/folders/1F5Qlyi30a3VNu9ZzCTKuTCDEmH6B4rdX</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>Spark® 20M multimode microplate reader</td><td>Tecan</td><td>N/A</td></tr><tr><td>PHERAStar® microplate reader.</td><td>BMG Labtech</td><td>N/A</td></tr><tr><td>Mithras LB 940 Multimode Microplate Reader</td><td>Berthold Technologies</td><td>N/A</td></tr></tbody></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Unless specified, HEK293 cells were used. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (ΔβArr1/2 and ΔQ-GRK cells) at 37°C and 5% CO2, and used up to passage 30. All cell lines were tested negative for Mycoplasma contamination.
## METHOD DETAILS

### Silencing of protein expression

Cells were plated at a density of 50000 cells/well and reverse-transfected with DharmaFECT1 Transfection reagent (Horizon), the plasmid encoding the GPCR under study and 50 nM of control-siRNA or 50 nM ON-TARGETplus Human AP2-μ2subunit. Experiments were performed 72 h after.

### DERET (internalization assay)

Cells were seeded at 50000 cells/well and reverse-transfected with lipofectamine and the targeted receptors labeled with a suicide enzyme in the N-terminal domain, either SNAP-, CLIP- or Halo-tag. Whereas otherwise noted, SNAP-tagged receptors have been used. 24 h after and prior to the experiment and for reducing the amount of ambient glutamate during the experiment cell medium was changed with medium containing GlutaMAX for 90 min at 37°C and 5% CO2.

Then, cells were labeled through the use of a specific substrate of the suicide enzyme that is tagging the GPCR. Cells were treated with either 100 nM of Tag-lite SNAP-Lumi4-Tb Labeling Reagent, 100 nM of Tag-lite CLIP-Lumi4-Tb or 100 nM of Tag-lite HaloTag-Lumi4-Tb Labeling Reagent for 1 h at 4°C in 1x Tag-lite SNAP/CLIP Labeling Medium (#LABMED, Revvity).

After labeling, Cells were washed with 100 µL of 1x Tag-lite SNAP/CLIP Labeling Medium (#LABMED, Revvity) twice and receptor expression was measured in Spark multimode microplate reader (Tecan). Compounds were then added at the indicated concentrations together with 25 µM fluorescein. The signal at 535 nm and 620 nm was recorded every 5 min during 60 min at 37°C in Spark multimode microplate reader (Tecan). In case of pharmacologically inhibiting the phosphorylation of GRK2/3 with cmpd101, after labeling with Tag-lite SNAP-Lumi4-Tb Labeling Reagent, cells were treated with 10 µM cmpd101 for 30 min at 4°C.

Kinetic data was expressed as internalization units in random fluorescent units (RFU), following the equation:

$$Internalization (RFU) = \frac{Ratio_{t=x} - Ratio_{t=0}}{Receptor expression} \cdot 1000000$$

where ratio refers to the emission at 620 nm (integrated during 1500 µs with a delay of measure of 1500 µs) over the emission at 535 nm (integrated during 150 µs with a delay of measure of 50 µs). Receptor expression refers to the emission signal at 620 nm before addition of fluorescein.

Data in bars was expressed as internalization units in RFU following the same formula as stated in kinetics data. Constitutive internalization was the internalization signal obtained after treating cells for 60 min with vehicle; agonist-induced internalization resulted from the signal obtained after agonist-treatment during 60 min minus the signal obtained for constitutive internalization. Otherwise noted, the vehicle condition is 100 µM LY341495, to avoid the effect of ambient glutamate.

### Recycling measurements (DERET)

Recycling measurements were DERET-based. HEK293 cells were plated at a density of 50000 cells/well and reversed lipofected with ST-mGlu5 or ST-mGlu3. After 24 h at 37°C and 5% CO2, medium was changed with medium containing GlutaMAX, and incubated 37°C and 5% CO2 for 90 min.

SNAP-tagged mGlu receptors were then labeled with 100 nM of Tag-lite SNAP-Lumi4-Tb Labeling Reagent in 1x Tag-lite SNAP/CLIP Labeling Medium for 1 h at 4°C. Then, the excess of labeling agent was washed with 100 µL 1x Tag-lite SNAP/CLIP Labeling Medium and receptor expression was measured in Spark multimode microplate reader.

Agonists were then added at the indicated concentrations together with 25 µM fluorescein, and incubated 60 min at 37°C and 5% CO2. After the induction of internalization by an agonist, the excess of agonist was simply washed (Wash) or a saturating concentration of LY341595 was added (100 µM LY34), together with 25 µM fluorescein. Internalization was measured at this time (time 0 min) and every 60 min after 4 h. Ago-internalization represent the specific internalization signal by subtraction of constitutive internalization. All recordings were done in Spark multimode microplate reader.

### TR-FRET for heterodimer detection

Cells were plated at a density of 100000 cells/well and reverse transfected with two different mGlu receptors, each tagged with a different suicide: either SNAP, CLIP or Halo. 24 h after, cells were incubated either with a Tag-lite SNAP-Lumi4-Tb Labeling Reagent, Tag-lite CLIP-Red Labeling Reagent (#SCLPREDF, Revvity) or Tag-lite HaloTag-Red Labeling Reagent substrate or a combination of both at a concentration of 100 nM, for 1 h at 37°C in 1x Tag-lite SNAP/CLIP Labeling Medium. Then, cells were washed 3 times with 100 µL 1x Tag-lite SNAP/CLIP Labeling Medium and TR-FRET was measured in PHERAStar microplate reader. Fluorescence was recorded with a delay of 60 µs and integrated over 400 µs, to be expressed the ratio 665 over 620, multiplied by 10000.

### BRET for βarr recruitment detection

Cells were plated at density of 100000 cells/well and reverse transfected: with the indicated GPCR tagged with a Venus protein for measuring direct recruitment. The indicated βarr tagged with RLuc8 was co-expressed in a proportion 3 to 1 (GPCR:βarr). After 24 h, the medium was changed with medium containing GlutaMAX for 90 min at 37°C. Cells were washed with PBS twice and incubated 10 min at 37°C. Receptor expression was measured in Mithras LB 940 Multimode Microplate Reader by measuring fluorescence
emitted at 535 nm after excitation at 485 nm. Then, 5 µM coelenterazine was added and BRET measurements were done during 500 s, before the indicated compounds were added. BRET signal was then measured for 1500 s miliBRET was calculated as the signal obtained at 535 nm over the signal obtained at 485 nm and adjusted by subtracting the ratios obtained when RLuc fusion proteins were expressed alone, and by multiplying the result by 1000. Data was corrected by the BRET signal obtained as a result of DOR-Venus recruitment of βarr, and represented then as % of βarr2 recruitment. Otherwise noted, the vehicle condition is 100 µM L341495, to avoid the effect of ambient glutamate.

## HTRF for endogenous β-arrestin2 recruitment

Cells were plated at a density of 100000 cells/well in 96 well plates and reverse-transfected with the plasmids encoding the GPCRs under study tagged with Venus fluorescent protein unless otherwise stated. 24 h later, the medium was changed with medium containing GlutaMAX for 90 min at 37°C. Cells were washed with PBS twice and incubated 10 min at 37°C. Receptor expression was measured in PHERAStar microplate reader at 520 nm emission and experiment was carried out following the specifications of Beta-Arrestin recruitment kit but with the only use of the antibody anti-beta arrestin 2 (Tb) at a concentration of 0.5 nM. After an incubation of 16 h at 22°C, HTRF ratio (520/620) was measured in PHERAStar FSX plate reader. Fluorescence was recorded with a delay of 60 µs and integrated over 400 µs, to be expressed the ratio 665 over 620, multiplied by 10000. Data was normalized as fold over basal (FOC over basal), were the agonist-induced signal was normalized over the control signal. Raw HTRF ratio of the control signal was represented as HTRF basal signal. Otherwise noted, the vehicle condition is 100 µM L341495, to avoid the effect of ambient glutamate.

## Western blot

Cells plated in 100 mm Petri dishes were washed once with ice-cold PBS and subsequently lysed with RIPA Buffer (ThermoFisher, 89900), supplemented with protease inhibitor cocktails (Merck, 5892953001). Cleared lysates were boiled with sodium dodecyl sulfate (SDS) loading buffer and 15 µg of total protein were loaded onto each lane of 8% polyacrylamide gels. After transfer onto nitrocellulose membranes, the total protein was detected by using specific antibodies (AP2: mouse anti-AP2 m2, BD Biosciences, 611351, (1:250), Actin: mouse anti-b actin, Sigma-Aldrich, A5441(1:5000)). As secondary antibodies, rabbit HTRF AbAnti-Mouse IgG d2-Conjugate (#61PAMDAA, Revvity) (1:100) was used.

## QUANTIFICATION AND STATISTICAL ANALYSIS

### Data analysis

All data analysis was performed using Prism 10 (GraphPad Software Inc., San Diego, CA, USA). Experiments were performed in triplicate, and final experiments represent the mean of at least three independent experiments, unless otherwise stated. Internalization kinetic data was adjusted to the pharmacokinetic equations described by Hoare S et al., 2020. Two models were compared: a “rise to steady state time course” fitting with a “baseline then rise to steady state time course”, based on the observation that the “rise to steady state time course” was not a well fit for every graph, particularly for ST-mGlu3. The comparison allowed to choose the fitting model by the use of an extra sum-of-squares F test. The relative amount of BRET was quantified as the ratio between Venus fluorescence and RLuc activity (×1000) and expressed as milli net BRET units.

### Statistical analysis

Statistical significance was assessed by a normality test (Shapiro-Wilk test) followed by (a) two-tailed Student's t test when comparing two groups; (b) one-way ANOVA and followed by a Dunnett's post-hoc test for comparing more than two groups; (c) a two-way ANOVA followed by a Šidák post-hoc test for multiple comparisons. Statistical significance was determined at *p* < 0.05. Asterisks indicate significance levels as follows: *p* < 0.05 (*), *p* < 0.01 (**), *p* < 0.001 (***), *p* < 0.0001 (****). Non significance, ns, was determined at *p* > 0.05