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Feeder or trigger – CCR2 as a scavenger and regulator of cell migration

Canonical chemokine receptors – scavenging “decoys”

Chemokine receptors coordinate cell migration upon interaction with their cognate chemokines. Upon activation, chemokine receptors coupe to the Gαi class of heterotrimeric G proteins, which, in turn, activate signaling pathways that ultimately lead to cell migration.

The chemokine receptor family comprises 19 canonical chemokine receptors together with 4 atypical chemokine receptors (ACKRs) which do not couple to G proteins and behave as scavenging “decoys” in order to either limit chemokines spatial availability or to remove them from in vivo sites, while maintaining the responsiveness of canonical G protein–coupled chemokine receptors that bind to the same ligand(s) (R. J. B. Nibbs et al. 2013). Although less characterized, canonical chemokine receptor systems have been also shown to not only directly regulatE migration but also play a scavenging role (e.g. CCR2, CXCR2, CXCR3, and CX3CR1) (A. E. Cardona et al. 2008). CCR2 is an example of a dual-function receptor that directly regulates both cell migration and scavenging (S. Volpe et al. 2012).

Molecular signature of CCR2 scavenging - no G proteins, no GRKs, no arrestins, and no clathrin

Chemokine scavenging mechanism involves internalization and recycling of the receptor with clearance of the ligand from the extracellular space however, the molecular cues involved in this process are poorly characterized. In this study, the molecular signature of CCR2 scavenging role is investigated. CCR2 canonical signaling requires the activation of Gαiβγ, followed by phosphorylation of the receptor C terminus by G protein receptor kinases (GRKs), specifically GRK2 and GRK3 (A. M. Aragay et al. 1998). Then β-Arrestin recruitment takes place allowing receptor internalization through clathrin-mediated pits (V. V. Gurevich, et al. 2018; Y. A. Berchiche et al. 2011). In this study, the role of these 3 molecular players (G proteins, GRKs, and β-arrestin) is investigated.

Removal of G proteins by using CRISPR KO of Gαi (Gαi KO) or KO of all Gα subtypes (Gα_all KO) (M. Grundmann et al. 2018), did not affect chemokine scavenging, consistent with prior work (S. Volpe et al. 2012; B. N. Zhao et al. 2019). Recruitment of both β-arrestin1 and β-arrestin2 was significantly diminished iin Gαi KO and Gα_all KO HEK293 cells, however there was only a minor loss in receptor internalization, which is surprising since internalization is known to be a consequence of β-arrestin recruitment. In addition, constitutive internalization of CCR2 was shown to be G protein-independent by a “prelabel” flow cytometry assay (B. N. Zhao et al. 2019) together with confocal fluorescence microscopy. Regarding GRKs role in CCR2 scavenging, BRET, flow cytometry and fluorescence microscopy readouts revealed that CCL2-dependent internalization was partially decreased in GRK2/3 KO cells and almost completely lost in the GRK2/3/5/6 KO cells, whereas constitutive receptor internalization was unaffected. Moreover, HEK293 cells with CRISPR KO of both β-arrestin1 and β-arrestin2 only led to a small but measurable loss of CCL2 scavenging and did not affect constitutive receptor internalization, with the same observations made for CCR2-expressing THP-1 monocytic cell line, together with an increase in migration, consistent with the role of β-arrestin in receptor desensitization. Although some GPCRs constitutively internalize through clathrin-coated pits independently of phosphorylation and β-arrestin (M. M. Paing et al. 2022; J. L. Parent, et al. 2001), inhibition of clathrin-mediated endocytosis (CME) did not affect the ability of CCR2 to scavenge chemokine and had only a small effect on constitutive internalization. Using fast recycling, slow recycling, and late endosomal recycling markers, CCR2 was also shown to be rapidly sorted into fast-recycling Rab4-positive endosomes in a more sustained manner compared to the atypical scavenging receptor ACKR3, and Rab7-positive late endosomes. CCR2 was also shown to be resistant to degradation and most likely recycling through the trans-Golgi network (TGN) where Rab7 is associated (A. Sapmaz et al. 2019, M. N. J. Seaman 2012). The ability to rapidly recycle, which was shown by a chemokine washout BRET assay (Y. Namkung et al. 2016), and avoid depletion may also contribute to its ability to efficiently scavenge chemokine.

CCR2 vs CCR1 scavenging signatures

CCR1 is constitutively phosphorylated, constitutively interacts with β-arrestin2, and constitutively internalizes in a β-arrestin2–dependent manner (C. T. Gilliland et. al 2013). In line, β-Arrestin KO cells showed a major reduction in CCL14 scavenging by CCR1, in contrast with a minor effect observed for CCR2. β-arrestin KO cells revealed the rapid reassociation of Gαi and Gβγ on CCR1, in contrast with CCR2, indicating that β-arrestin regulates scavenging and signaling of CCR1 to a greater extent than CCR2.

Functional relevance and implications of scavenging of canonical chemokine receptors

Clearing circulating chemokines and chemokines from tissue microenvironments has been well described for ACKRs which work in a team with canonical chemokine receptor to drive regulated immunological responses and inflammatory conditions, preventing overstimulation and activation. However, this scavenging role has been also described for several canonical chemokine receptors, including CCR2.

In this study, it is revealed that CCR2 scavenging role was largely independent of the classical GPCR signaling pathways, revealing the existence of two distinct functional populations – one that directs cell migration and one that regulates scavenging. But why do canonical chemokine receptors have this dual role in physiology? Scavenging may allow cells to continuously migrate by remaining responsive to chemokines (S. Volpe et al. 2012); it dampens the inflammatory response when needed (C. A. H. Hansell et al. 2011); and it may interfere with other chemokine receptors which share the ligands and affect cell migration (A. E. Cardona et al. 2008). However, it is important to note that this scavenging role does not occur in all chemokine receptor systems such as CXCR4, which relies on ACKRs. How balanced is this dual role? In monocytes and dendritic cells exposed to treatments mimicking inflammation, CCR1, CCR2, and CCR5 switch purely to scavenging (G. D’Amico et. al 2000), becoming incapable of promoting cell migration, a phenomenon which is likely to be mediated by changes in the cell motility machinery with receptor-specific switches not yet described to play a role.

What are the implications in drug discovery? This scavenging function should be considered when evaluating the safety and therapeutic efficacy of blocking receptor-ligand binding. CCR2 inhibition leads to inhibition of scavenging and elevated plasma levels of CCL2 (Y. Wang et al. 2009; R. J. Aiello et. al 2010) which may ultimately compete with receptor antagonists, thereby decreasing the efficacy (J. Gilbert et. al 2011). This study provides a comprehensive analysis of CCR2 scavenging role however to fully understand the role of canonical chemokine receptors scavenging population pathophysiology, a better understanding of the regulatory mechanisms will be required.

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