# Chemokine receptors – Exemplifying functional divergence in G protein-coupled receptors

Omolade Otun, Sébastien Granier, Thierry Durroux, Cherine Bechara

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Omolade Otun, Sébastien Granier, Thierry Durroux, Cherine Bechara. Chemokine receptors – Exemplifying functional divergence in G protein-coupled receptors. Molecular Pharmacology, 2025, 107 (7), pp.100053. 10.1016/j.molpha.2025.100053. hal-05173705

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Chemokine Receptors – Exemplifying functional divergence in
GPCRs

Omolade Otun¹*, Sébastien Granier, Thierry Durroux and Cherine Bechara¹,²*

¹Institut de Génomique Fonctionnelle, University of Montpellier, CNRS, INSERM, 34094 Montpellier Cedex 5, France

²Institut Universitaire de France, Paris, France

*Corresponding authors: o.o.otun@vu.nl and cherine.bechara@igf.cnrs.fr
Significance Statement

This mini-review explores how signalling in the chemokine receptor family diversifies at the ligand, transmembrane, and intracellular levels. This functional diversity presents unique opportunities for drug development by selectively targeting distinct pathways.
Abstract

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G protein-coupled receptors are known for their ability to generate a wide range of functional
responses through interaction with various intracellular partners. This signalling versatility is
particularly evident within the chemokine receptor family, where conventional receptors signal
primarily through classic G protein-coupled pathways whilst atypical chemokine receptors
appear not to possess such ability but instead couple to alternative intracellular partners such as
β-arrestins. Functional diversity in signalling presents unique opportunities for drug
development, allowing different pathways to be selectively targeted to meet specific therapeutic
needs. This mini-review explores the mechanisms by which GPCR signalling, particularly
within the chemokine receptor family, can be diversified at the modulatory, transmembrane and
intracellular level.
Introduction

G protein-coupled receptors (GPCRs) are heptahelical proteins and represent the largest family of membrane proteins (Zhang et al., 2024). Responsible for mediating a variety of physiological processes, they play a central role in maintaining cellular communication and homeostasis. Their importance is reflected in their pharmaceutical relevance, with over 30% of FDA-approved drugs targeting GPCRs (Hauser et al., 2017). GPCR signalling is a complex process, diversified at several levels including modulation, receptor conformations and their interactions with intracellular partners as well as other GPCRs. At the modulatory level, ligands induce specific conformations in GPCRs, enabling diverse signalling pathways. By acting as guanine nucleotide exchange factors (GEFs), activated GPCRs undergo conformational changes leading to the recruitment of G protein heterotrimers and the subsequent activation of subtype-dependent signalling cascades. The functional output of GPCRs is further diversified by their ability to signal in a G protein-independent manner, engaging GPCR kinases (GRKs) and arrestins (Gurevich & Gurevich, 2019). Consequently, ligands can exhibit biased agonism, preferentially activating specific signalling pathways while bypassing others (Kolb et al., 2022). This phenomenon creates challenges for drug discovery as certain pathways can be linked to therapeutic or adverse effects. Nonetheless, biased ligands present significant therapeutic potential, allowing for the selective targeting of beneficial pathways while minimizing unwanted effects. For example, TRV130, a G protein-biased agonist at the μ-opioid receptor has been shown to induce fewer side effects despite displaying analgesic efficacy comparable to that of Morphine (Altarifi et al., 2017; DeWire et al., 2013). Similarly, biased activation of the β-arrestin pathway through the Angiotensin II type 1 receptor has exhibited several cardioprotective effects (Garland & Vuylsteke, 2020; Singh & Karnik, 2024).

Amongst GPCRs, the Chemokine Receptor (CR) family represents a good model for investigating functional divergence. CRs play a critical role in immune regulation, orchestrating
both inflammatory and homeostatic immune responses (Hughes & Nibbs, 2018). They elicit
their effects upon binding to small 8 kDa proteins known as chemokines. This system exhibits
promiscuity in that several chemokines can bind to the same receptor and a single chemokine
can bind to multiple receptors which is likely contributing to the broad functional diversity of
CRs (Szpakowska et al., 2023). Whilst majority of CRs are Classical Chemokine Receptors
(CCRs) in that they signal predominantly through Gαi/o family of G proteins, a small subset of
Atypical Chemokine Receptors (ACKRs) appears to be unable to. This quality has led to
ACKRs largely being described as scavengers due to their high affinity for chemokines and
lack of G protein signalling (Ulvmar et al., 2011). This has been thoroughly challenged, with
evidence showing that ACKRs possess their own signalling mechanisms through alternative
pathways, further broadening the signalling outputs of the chemokine receptor network
(Borroni et al., 2013; Le Mercier et al., 2021; Matti et al., 2020; Rajagopal et al., 2010).

This diversification of GPCR signalling highlights the adaptability of these receptors in fine-
tuning various physiological processes. Understanding this diversity presents opportunities to
explore novel therapeutic targets. This mini-review aims to address some of the mechanisms
by which GPCR signalling can be diversified within the CR system at the level of the
modulators, intrinsic receptor properties, and intracellular partners (Fig. 1).

Diversifying GPCR signalling at the modulatory level

A large network of promiscuous chemokines

Chemokines are highly conserved in their structure and can be classified into four subgroups
determined by the arrangement of conserved cysteines located at their N-termini, these being
C-, CC-, CXC- and CX₃C (Miller & Mayo, 2017). With the chemokine network consisting of
45 chemokines and 24 CRs (Szpakowska et al., 2023), multiple chemokines can bind to the
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same receptor, and individual chemokines can interact with multiple receptors, resulting in the CR system being described as promiscuous. Consequently, debate exists as to whether this represents a redundancy within this system as this overlap would suggest that if one chemokine pathway is blocked, another can compensate to maintain essential immune functions. This has been proposed to be the main reason for the failure of drugs targeting the chemokine network in inflammatory diseases (Dyer, 2020). However, some have argued that this redundancy may be absent in a more physiological setting, where contextual factors could diversify signalling ways that are difficult to replicate *in vitro*. This has been explored particularly for CXCL9, CXCL10 and CXCL11 chemokines binding CXCR3 (Christensen et al., 2006; Groom & Luster, 2011). Further investigation into these factors may provide insight into how chemokine signalling is refined *in vivo*, enhancing our understanding of this network.

Non-chemokine ligands

In addition to chemokines, other natural molecules have been shown to bind CRs. Macrophage migration inhibitory factor (MIF) is one such ligand, an inflammatory cytokine that exerts chemokine-like properties through atypical interactions with CXCR2, CXCR4 (Bernhagen et al., 2007) and ACKR3 (Alampour-Rajabi et al., 2015). For ACKR3 in particular, several non-canonical ligands have been detailed. For example, ACKR3 has been recently described as a receptor for opioids, able to regulate their circulating concentrations and dampening the available pool for opioid receptors (Meyrath et al., 2020). Another ligand for ACKR3 is derived from the cleavage of the precursor peptide proadrenomedullin, which generates proadrenomedullin N-terminal peptide (PAMP), an agonist that also activates ACKR3 (Meyrath et al., 2021).

CRs can also be targeted by pathogens such as bacteria, viruses and parasites (Alcami & Lira, 2010; Chensue, 2001; Kraemer et al., 2022). For example, the HIV envelope protein gp120
binds CXCR4 and CCR5 using them as co-receptors for viral entry (Bleul et al., 1996; Deng et al., 1996). Another example is the family of pore-forming bicomponent leukotoxins, virulence factors produced by *Staphylococcus Aureus* which lyse specific cells upon binding to CRs (Seilie & Bubeck Wardenburg, 2017). Though little is known regarding their impact on receptor downstream signalling, their binding was shown to impact the intracellular conformation of ACKR1 as well as receptor-receptor interactions (Grison et al., 2021). In addition, several structural studies have highlighted regions that may facilitate interactions between these toxins and specific CRs, such as the presence of sulfated tyrosine residues in receptor N-termini (Lambey et al., 2022; Spaan et al., 2015), a feature that is prevalent amongst CRs (Ludeman & Stone, 2014). These findings highlight the broad ligand-binding capabilities of CRs that may underscore the complexity of their action in vivo.

Chemokine oligomerisation

Adding another layer of complexity to the chemokine network is the fact that chemokines can readily form homo- and hetero-oligomers. Chemokines CCL5, CXCL1, CXCL8 and CXCL12 are just some examples that exist both as monomers and dimers under physiological conditions (Horcher et al., 1998; Ravindran et al., 2013; Ray et al., 2012). Chemokine dimerization can be induced by their binding to glycosaminoglycans (GAGs) which is essential for their ability to induce leucocyte migration *in vivo* (Proudfoot et al., 2003). This oligomerization appears to be necessary for GAG binding, as it exposes new GAG binding sites (Dyer et al., 2015; Liang et al., 2016). Moreover, GAG binding specifically promotes formation of higher order oligomers of chemokines (Hoogewerf et al., 1997). This propensity allows regulation of the chemokine system by limiting the concentration of circulatory chemokines and instead creating a high local concentration along which leucocytes can migrate (Blanchet et al., 2023).
Two modes of dimerization were observed that distinguish CC- and CXC- chemokine classes.

The interface of CC chemokine dimers involves the structuring of N-termini of both protomers to form a 2-stranded anti-parallel β-sheet. Some CC dimers have been shown to be inactive (Jin et al., 2007; Tan et al., 2012) which may be attributed to the important role the N-terminus plays in CR activation. On the other hand, CXC chemokines dimerise via their β1 strand (Jansma et al., 2010) creating a six-stranded β sheet. This leaves the chemokine N-terminus free for receptor activation. The functionality of these oligomers has been explored on several levels.

Studies on CXC chemokine dimers have used locked dimers stabilized by disulfide bonds introduced at the dimer interface. These studies have shown that chemokine dimers retain the ability to bind their receptors whilst exhibiting distinct pharmacological properties compared to monomers (Drury et al., 2011; Ravindran et al., 2013; Tan et al., 2012). For example, CXCL12 dimers can bind and internalise via ACKR1, a receptor not recognized by monomeric CXCL12 (Gutjahr et al., 2021). The diverse range of ligands and their ability to adopt different structures—particularly through oligomerization, which regulates their local and global concentrations—offer valuable insights into the regulation of physiological functions. This also raises important structural questions about receptor's ability to recognise such a broad spectrum of ligands and its capacity to activate multiple signalling pathways.

## Diversifying GPCR signalling at the receptor level

Class A GPCRs accommodate a diverse range of ligands through structural variations in the size, shape, and electrostatic properties of the binding pocket and extracellular loops (ECLs) (Katritch et al., 2012). The chemokine-CR system reflects this, as CRs exhibit broad structural diversity at their extracellular face. GPCR functional diversity arises from structural features that both influence and are influenced by ligand binding, receptor conformational changes and
oligomerization. These factors collectively shape receptor functionality, contributing to diverse
signalling outcome.

Within the chemokine network, one manifestation of this diversity is the intrinsic signalling bias of ACKRs. ACKRs 2–5 predominantly signal through β-arrestins (Borroni et al., 2013; Le Mercier et al., 2021; Matti et al., 2020; Rajagopal et al., 2010) and are thus classified as intrinsically biased receptors. This raises the question of which structural features confer this behaviour to ACKRs, given their close structural homology to CCRs (Aleotti et al., 2024). Understanding these features could provide valuable insights for biased drug development.

## Ligand binding mechanisms

Ligand interactions at GPCR binding pockets have been proposed to contribute to diverse
signalling. Initial crystal structures of rhodopsin revealed that GPCRs feature a main (major)
pocket formed by TMs 3, 4, 5, and 6, and a shallower (minor) pocket composed of TMs 1, 2,
3, and 7 (Rosenkilde et al., 2010; Schwartz et al., 2006). While most ligands for class A GPCRs
bind predominantly within the major pocket, chemokines and other endogenous peptides
including apelin and neurotensin can engage the major and minor pockets concomitantly when
binding to their respective GPCRs (Vass et al., 2018). This dual engagement of both sub-
pockets exhibited by chemokines suggests that developing drugs aimed at either pocket may
provide new insights into receptor behaviour and the mechanisms of diverse signalling.

Chemokine-CR binding was initially described to occur as a 2-step process. In the first step, the
receptor's N-terminus, representing chemokine recognition site (CRS) 1, binds to the
chemokine site 1 (CS1) at the level of the chemokine's core. This orients the ligand so that its
N-terminus (CS2) binds to the receptor's orthosteric pocket involving the TMs and ECLs
(CRS2) leading to receptor activation (Crump et al., 1997). This model has since been shown
to be an oversimplification. With an increase in the number of high-resolution structures of
chemokine-CR complexes, chemokine binding is recognised more as a multi-step and multi-site process (Kleist et al., 2016) with CRS1.5 and CRS3 serving as additional conserved binding features. Most of these structures have shown commonalities in which the CR N-terminus wraps the chemokine core whilst the chemokine N-terminus binds the orthosteric pocket.

ACKR3 was the first ACKR for which a high-resolution structure was resolved (Yen et al., 2022), revealing significant differences compared to the structures of CCRs. Notably, CXCL12 is rotated by 70-80° in ACKR3 relative to its orientation in CXCR4 (Liu et al., 2024; Yen et al., 2022) (Fig. 1, inset). This repositioning enables the ACKR3 N-terminus to contribute an additional β-strand to the CXCL12 β-core, a feature that has not been observed in CCRs. However, CCL7 did not show the same β-strand contribution to ACKR1 (Saha et al., 2024), therefore an altered mode of engagement at the receptor N-terminus cannot be regarded as a universal feature of ACKRs binding to chemokines.

The N-terminus has also been proposed to confer atypical behaviour to ACKRs due to the pharmacological properties of CXCR3 isoforms. Compared to the canonical form CXCR3-A, the splice variant CXCR3-B possesses a long N-terminal region (Ehlert et al., 2004; Lasagni et al., 2003). These isoforms were shown to differ in their preferred signalling pathways and CXCR3-B has been shown to signal in a manner similar to ACKRs. Indeed, unlike CXCR3-A, CXCR3-B does not couple to G proteins and signals predominantly through β-Arrestin (Smith et al., 2017). As such, CXCR3-B has been found to be predominantly intracellular (D’Uonnolo et al., 2022). Interestingly, gradual N-terminal truncation of CXCR3-B reinstated its G protein signalling ability (D’Uonnolo et al., 2022), further suggesting that the N-terminus may play a role in atypical behaviour.
## The DRY motif

The DRY motif, located at the intracellular end of TM3, is one of the most conserved features of class A GPCRs (Rovati et al., 2007). In particular, R<sup>3.50</sup> has been demonstrated to interface with both G proteins and arrestins in high-resolution structures of GPCR complexes (Hauser et al., 2021). Since the majority of ACKRs, except ACKR3, presents alterations or absence of the DRY motif (Cancellieri et al., 2013), it is rational to expect that these deviations confer atypical properties leading to the lack of G protein signalling. However, this hypothesis is challenged since CCRs CXCR6 and XCR1 have a modified DRY motif yet signal predominantly through Gαi (Chandrasekar et al., 2004; Fox et al., 2019). ACKR3 represents a special case, since it generally does not activate Gαi, except in some cellular contexts (see below), yet it does contain a DRY motif. Our group has shown that various small-molecule ligands induced a decreased accessibility of the DRY motif which becomes shielded by TM6 in the presence of inverse agonists with regards to β-arrestin recruitment, or by TM5 and Y257<sup>6.40</sup> in the presence of an agonist (Otun et al., 2024). Shielding of the DRY could be related to the inability of ACKR3 to activate G proteins. Finally, several groups have attempted to replace the ACKR3 ICL2 region proximal to the DRY with the ICL2 of a CCR in order to restore G protein signalling ability but these attempts have been unsuccessful (Hoffmann et al., 2012; Naumann et al., 2010; Yen et al., 2022), reinforcing the hypothesis that multiple structural elements may contribute to the lack of G protein signalling by ACKRs.

## Receptor oligomerisation

Signalling potential of CRs is further expanded by their ability to form higher-order oligomers, similar to their ligands. Oligomerisation of GPCRs has long been proposed as another mechanism of GPCR regulation at the receptor level (Gomes et al., 2016). There is increasing evidence to show that CRs can assemble into both homo- and heterodimers with unique functional properties, and that dimerization could be modulated by cognate chemokines and
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small ligands. For instance, CCL2 was able to induce homo-dimerization of CCR2 (Rodríguez-Frade et al., 1999) and CXCR4 (Vila-Coro et al., 1999) respectively, whilst IT1t, a small ligand inverse agonist for G protein recruitment, was shown to decrease the dimeric state of CXCR4 (Işbilir et al., 2020; Ward et al., 2020). This was also recently demonstrated for CCR3 as chemokines CCL11, CCL24, CCL5 and CCL8 were shown to induce receptor oligomerisation to varying degrees (Li et al., 2024). The impact of oligomerisation on receptor's function has been most extensively studied for CXCR4. Its initial homodimeric structures (Wu et al., 2010) revealed distinct dimer interfaces depending on whether CXCR4 was bound to the cyclic peptide CVX15 or to the small ligand inverse agonist IT1t. In the case of CVX15, the dimer interface was stabilised by TM3, TM4 and ICL2 with contacts present on both the extracellular and intracellular faces. In contrast, the IT1t-bound dimer interface involved only TM5 and TM6, with contacts limited to the extracellular face. The resolution of CXCR4 trimeric and tetrameric structures has given further insight into how GPCR oligomerisation may receptor functionality (Saotome et al., 2024). Both trimers and tetramers of CXCR4 formed similar interfaces with TM5, TM6, and TM7 of one protomer interacting with TM1 and TM7 of the neighbouring one. Despite these similarities, the intracellular region showed remarkable differences in that tetrameric CXCR4 showed a sterol-driven displacement of TM6 relative to the trimeric conformation, enabling therefore the rotation of the subsequent protomer. Saotome et al. suggest that this allows the trimeric conformation to retain an active state cavity to which the α5-helix of the Gα subunit can bind whilst the tetrameric state would sterically hinder this binding. This proposes a mechanism through which oligomeric assembly further expands the GPCR signalling repertoire.

Some ACKRs have also been proposed to exert their regulatory effects through heterodimerisation with CCRs. ACKR3 was shown to co-immunoprecipitate with CXCR4 (Décaillot et al., 2011) and co-expression of the two receptors impaired CXCR4 signalling at
the level of Ca²⁺ mobilisation and Gαi activation (Levoye et al., 2009). Similarly, co-expression of ACKR1 with CCR5 showed that the two can form a heterodimer that induced robust reduction in chemotaxis and CCR5-mediated signalling but maintained affinity of CCR5 for CCL5 (Chakera et al., 2008). In addition, chemokine receptors can also heterodimerise with non-chemokine receptors as has been suggested to occur with the cannabinoid (Coke et al., 2016) and opioid receptors (Chen et al., 2004; Parenty et al., 2008; Pello et al., 2008). Together, these findings highlight heterodimerisation as a mechanism through which chemokine receptors can modulate each other's signalling, either by dampening responses or altering ligand affinity or functionality.

# Diversifying interactions between GPCRs and their intracellular partners

## Receptor phospho-barcoding by GRKs

GRKs play an integral role in fine-tuning cellular responses initiated by GPCR activation and are important in regulating GPCR desensitisation (Gurevich & Gurevich, 2019). Though multiple kinases are able to act on GPCRs, GRKs are considered as the main drivers of GPCR phosphorylation, which is a pre-requisite for their subsequent recruitment of β-arrestins (Smith & Rajagopal, 2016). Different GRKs have been shown to create distinct phosphorylation 'barcodes' which determine the interaction between the GPCR and arrestin and possibly the subsequent signalling cascade (Liggett, 2011; Nobles et al., 2011). For instance, the regulatory ability of receptor's barcoding events has been demonstrated for CXCR3 which presented different phosphorylation barcodes at the C-terminal tail depending on the bound chemokine (Eiger et al., 2023). This was shown to be potentially linked to the actions of GRKs as they also found differences in the degree to which specific phosphodeficient CXCR3 mutants can recruit GRKs 2 and 3 upon stimulation with the various chemokines. Disruption of these barcodes also
differentially impacted G protein and β-arrestin signalling of each chemokine at CXCR3.

Interestingly, phospho-deficient CXCR4 mutants affected the conformation of receptor-bound β-arrestin. This would thus indicate that induction of specific phosphorylation patterns by different chemokines causes different conformations of β-arrestin which may have downstream consequences. Indeed, it was found that phospho-deficient mutants also affected more distal consequences of CXCR3 signalling such as T-cell chemotaxis (Eiger et al., 2023). This highlight both the regulatory role that GRK phosphorylation play in GPCR signalling and the non-redundancy in the chemokine network.

Experiments performed with GRK knockout cell lines have begun to determine the reliance of GPCRs on different GRKs and how this correlates to their ability and preference in recruiting either β-arrestin isoform (Drube et al., 2022; Pandey et al., 2021). For example, Pandey *et al.* highlighted that CCR2 and ACKR2 differed significantly in their reliance on GRKs for β-arrestin recruitment, with GRK5/6 knockout abolishing arrestin recruitment to CCR2 but enhancing it for ACKR2 upon CCL7 binding (Pandey et al., 2021). Another study also investigated the ability for ACKR3 to recruit GRKs showing that ACKR3 recruits GRKs 2, 3 and 5 to varying degrees whilst being unable to recruit GRK6 (Zarca et al., 2021). Given the importance of GRK phosphorylation in facilitating the binding of arrestins, one could speculate that targeting GPCR signalling at this level may be imperative to influence bias towards or against arrestin binding.

## Versatile interactions with β-arrestin

Structural studies captured two modes of interaction between GPCRs and arrestins. The ‘core conformation’ in which the arrestin finger-loop binds to the cytoplasmic cavity of the receptor or a ‘hanging’ conformation in which β-Arrestin binds to the receptor only by the phosphorylated C-tail (Shukla et al., 2014). The different conformations have been shown to have distinct effects with regards to arrestin activation and subsequent cellular effects. Studies
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investigating the signalling properties of core- and hanging-only β-arrestin mutants showed that both conformations can initiate receptor internalisation. One main difference is that the core conformation completely occludes the G protein binding interface causing desensitisation whilst it has been shown that the hanging conformation allows for sustained G protein signalling (Cahill et al., 2017; Haider et al., 2022). Recently, the cryo-EM structures of ACKR3 in complex with β-arrestin1 and 2 were published as a preprint showing that the arrestin finger loop did not engage the receptor core unlike the majority of GPCR-arrestin complexes and instead adopted a hanging conformation for GRK2 phosphorylated receptors (Q. Chen et al., 2023). This differential mode of engagement was proposed to contribute to ACKR3s non-canonical behaviour. It was further highlighted that GRK2- or GRK5-driven phosphorylation barcodes at the C-tail of ACKR3 play a role in determining the arrestin interface at ACKR3.

The versatility of the interaction with β-arrestin was also demonstrated in our recent work in which we used Hydrogen-Deuterium exchange mass spectrometry analysis to monitor the binding interfaces between ACKR3 and β-arrestin 1 lacking the auto-inhibitory C-terminal region (Otun et al., 2024). This work revealed that binding of β-arrestin1 induced stabilising effects at ACKR3 all ICLs, the most impacted being ICL2, which may indicate direct engagement of all ICLs by β-arrestin. As the different studies performed to monitor ACKR3/ β-arrestin1 binding differed in terms of added ligand, receptor phosphorylation states, and the presence of absence of a lipid bilayer, this underscores the versatile and dynamic nature of the system, highlighting how different cellular contexts can lead to varying insights into GPCR interaction with arrestins.

## Versatile interactions with G-proteins

It is generally accepted that, unlike CCRs, ACKRs do not activate G proteins. However, this notion has been challenged by a handful of publications reporting evidence of G protein activation or recruitment under certain conditions. For instance, some reports demonstrated that
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ACKR3 activates Gαi in astrocytes and glial cells, eliciting responses similar to those induced by CCRs such as cell proliferation, migration and ERK phosphorylation (Fumagalli et al., 2020; Ödemis et al., 2012), while others have shown that ACKR3 can bind to Gα subunits without activation (Levoye et al., 2009). Another example is ACKR1, which was shown to interact with Gαi constitutively, an interaction that was inhibited upon ACKR1 binding to S. aureus leukotoxins at the extracellular level (Grison et al., 2021). These findings raise the question of whether ACKRs and other GPCRs might exhibit diverse mechanisms of G protein engagement beyond traditional models.

Non-canonical modes of interaction have indeed been observed for certain GPCRs in complex with G proteins (Feng et al., 2023; Kato et al., 2019; Yue et al., 2022). The Neurotensin Receptor 1 (NTSR1) is an example for which high resolution structures of both canonical and non-canonical engagement has been described (Kato et al., 2019). Whilst the canonical state showed features of a fully activated receptor with respect to conformations of conserved microswitches, the non-canonical state showed features of both active and inactive conformations. Since the non-canonical state could not induce G protein activation, it was suggested to represent an intermediate state within the activation process as demonstrated by molecular dynamics simulations (Kato et al., 2019). These findings reinforce the notion of intermediate conformations that add nuance to GPCR signalling outputs by enabling a spectrum of activation states. This raises the possibility that the relationship between ACKRs and G proteins may involve similar non-canonical interactions, prompting further questions about the physiological roles of such interactions.
Conclusion

The functional diversity exhibited by GPCRs offers great therapeutic potential and is well-exemplified in the CR family. In this minireview, we have explored some of the mechanisms that diversify CR signalling, including ligand promiscuity, receptor oligomerisation, intracellular interactions, and the unique behaviours of ACKRs. Understanding the mechanisms through which GPCR signalling is regulated paves the way to target GPCR signalling without targeting the receptor itself. For instance, the ability of chemokine receptors to bind diverse ligands, from small-molecules to chemokines to bacterial proteins, provides valuable tools to understand the processes underlying ligand recognition. Furthermore, expanding the range of tools available to investigate CRs could significantly enhance our ability to explore the more elusive aspects of their signalling. This includes uncovering non-canonical signalling pathways of ACKRs and resolving questions about whether the CR system exhibits specificity or redundancy. Moreover, studies with locked chemokine dimers have demonstrated their influence on CR signalling, affecting both dimer pharmacology and chemokine availability via interactions with GAGs. The physiological role of chemokines and CR dimers remains poorly understood. There remains the possibility that targeting chemokine or CR dimers, rather than monomers, could represent a novel and underexplored therapeutic strategy.

The interactions between CRs and their intracellular partners is one example that underscores
signalling diversity within the CR family, particularly with respect to ACKRs. The observation
that ACKR3 can activate Gαi in astrocytes (Fumagalli et al., 2020; Ödemis et al., 2012) is
particularly interesting as it indicates potential context-dependent function of the CR system.
The impact of the cellular context on CR signalling was demonstrated in a recent study
demonstrating how anionic lipids enhance the affinity of chemokines for ACKR3 (Eberle &
Gustavsson, 2024). Such findings emphasize the need to select appropriate cellular models for
*in vitro* studies, as these models significantly impact our ability to accurately understand the
nuances of CR signalling. They also highlight the need to suggests that all the signalling
mechanisms elucidated in heterologous expression systems should be validated in physiological
conditions. In conclusion, CRs are able to diversify their signalling using mechanisms
employed by many GPCRs. Understanding and leveraging these mechanisms will give great
insights into innovative therapeutics towards GPCRs.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Overview of features diversifying chemokine receptors signalling. Binding partners exhibited by chemokine receptors, including chemokines and their dimers, small-molecule ligands such as opioids, and pathogen-derived molecules. Inset shows the comparison of CXCL12 binding modes on CXCR4 (PDB: 8K3Z) and ACKR3 (PDB: 7SK3), the N-terminus of ACKR3 contributing an additional beta strand to CXCL12. CXCL12 is shown in light pink, CXCR4 in pale green and ACKR3 in light blue. At the level of the membrane, chemokine receptors have the potential to oligomerise with other chemokine and non-chemokine receptors (double sided arrows) which may lead to crosstalk involving potentiation or inhibition of canonical signalling. Here, specific lipids (depicted in orange and soft pink) could play a role in stabilising receptor-receptor interactions. Finally, binding and/or activation of intracellular partners could be also diverse: different GRKs induce distinct phosphorylation patterns at receptor's C-termini, presenting a phosphorylation barcode that may be differentially recognised by β-arrestin subtypes, whereas some receptors are capable of activating G proteins and others can only recruit G proteins without activation.</figcaption></figure>
## Acknowledgments

Authors acknowledge funding by the European Union (H2020-MSCA Program, Grant agreement 860229-ONCORNET2.0) and the French Agence Nationale de la Recherche (projects LEUKOCEPTOR ANR-21-CE44-0007-01 and OCHRE n° R19168LL).

## Data availability

This article contains no datasets generated or analyzed during the current study.
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