Multiple Roles for the C-terminal Tail of the Chemokine Scavenger D6

Abstract

D6 is a heptahelical receptor that suppresses inflammation and tumorigenesis by scavenging extracellular pro-inflammatory CC chemokines. Previous studies suggested this is dependent on constitutive trafficking of stable D6 protein to and from the cell surface via recycling endosomes. By internalizing chemokine each time it transits the cell surface, D6 can, over time, remove large quantities of these inflammatory mediators. We have investigated the role of the conserved 58-amino acid C terminus of human D6, which, unlike the rest of the protein, shows no clear homology to other heptahelical receptors. We show that, in human HEK293 cells, a serine cluster in this region controls the constitutive phosphorylation, high stability, and intracellular trafficking itinerary of the receptor and drives green fluorescent protein-tagged ␤-arrestins to membranes at, and near, the cell surface. Unexpectedly, however, these properties, and the last 44 amino acids of the C terminus, are dispensable for D6 internalization and effective scavenging of the chemokine CCL3. Even in the absence of the last 58 amino acids, D6 still initially internalizes CCL3 but, surprisingly, exposure to ligand inhibits subsequent CCL3 uptake by this mutant. Progressive scavenging is therefore abrogated. We conclude that the heptahelical body of D6 on its own can engage the endocytotic machinery of HEK293 cells but that the C terminus is indispensable for scavenging because it prevents initial chemokine engagement of D6 from inhibiting subsequent chemokine uptake.

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

“…In this study, we demonstrate for the first time that stimulation of CCX-CKR with CCL19, CCL21, and CCL25 indeed induced ␤-arrestin2-GFP translocation. CCX-CKR did not translocate ␤-arrestin2-GFP in the absence of chemokines, which is in contrast to the constitutively phosphorylated chemokine decoy receptor D6 (21). Moreover, fluorescently labeled CCL19 co-localized in the same intracellular vesicles as ␤-arrestin2-GFP, suggesting recruitment of ␤-arrestin to CCL19-bound CCX-CKR.…”

“…D6 constitutively recruits ␤-arrestin, which is essential for the observed continuous internalization of D6 (20). However, a more recent study provided evidence that ␤-arrestin was not necessarily involved in D6 internalization but increased receptor stability (21). CCX-CKR has been previously suggested to internalize CCL19 in a ␤-arrestin-independent manner (10).…”

D6 and the atypical chemokine receptor family: Novel regulators of immune and inflammatory processes

“…Importantly, the constitutive internalisation and recycling of D6 is associated with, although apparently not dependent on, ligand-independent constitutive phosphorylation of the C-terminal portion of the molecule [55,59]. Removal of the most C-terminal 44 amino acids, or mutation of 6 serine residues in this region, results in a block in phosphorylation but surprisingly has little effect on ligand internalisation by D6 [59]. It is still not clear why such large amounts of D6 are present within the cells with only relatively low representation on the cell surface.…”

“…We, and others, have demonstrated that D6 binds ligand at the cell surface and then internalises it in a Rab5-dependent manner via the recycling endosomal system before depositing it in acidic lysosomes for intracellular degradation [56,57]. Indeed D6 is capable of constitutively internalising and recycling, a process that may be dependent on b-arrestin [58] although the precise role for arrestins in the function of D6 remains to be resolved [59]. Importantly, the constitutive internalisation and recycling of D6 is associated with, although apparently not dependent on, ligand-independent constitutive phosphorylation of the C-terminal portion of the molecule [55,59].…”

“…Indeed D6 is capable of constitutively internalising and recycling, a process that may be dependent on b-arrestin [58] although the precise role for arrestins in the function of D6 remains to be resolved [59]. Importantly, the constitutive internalisation and recycling of D6 is associated with, although apparently not dependent on, ligand-independent constitutive phosphorylation of the C-terminal portion of the molecule [55,59]. Removal of the most C-terminal 44 amino acids, or mutation of 6 serine residues in this region, results in a block in phosphorylation but surprisingly has little effect on ligand internalisation by D6 [59].…”