β-Arrestin-dependent Constitutive Internalization of the Human Chemokine Decoy Receptor D6

Abstract

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor ␤-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with ␤-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from ␤-arrestin activate any Gprotein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.Leukocyte recruitment into inflamed tissues is mediated by chemokines acting on a distinct subfamily of G-protein-coupled receptors (GPCR). 1 This includes 18 receptors with the ability to activate G ␣i -protein-dependent signaling events and cell migration (1-4). Two other chemokine binding molecules, with homology to chemokine receptors but defective in signaling function, called Duffy antigen and D6, have been identified (5-7) and recently classified as "silent" chemokine receptors (8). Evidence in in vitro models, gene-targeted mice, and individuals with erythrocyte-restricted deficiency suggests that the Duffy antigen silent receptor may facilitate chemokine transport across endothelial cells as well as acting as a chemokine buffering and scavenging system (5, 9 -12). D6 is highly expressed in endothelial cells lining afferent lymphatic vessels (13) and supports rapid internalization and degradation of inflammatory CC-chemokines, acting as a chemokine scavenger (7). D6 was suggested to act as a gatekeeper on afferent lymphatic endothelium, preventing excessive transfer to lymph nodes of inflammatory chemokines and disruptive leukocyte recruitment (7). The molecular mechanisms underlying this scavenging function are unknown.Upon agonist binding, most GPCRs activate a signaling cascade mediated by G-protein activation leading to receptor phosphorylation by a G-protein-coupled receptor kinase, which results in uncoupling of the receptor from G-proteins (14). This desensitization process is further facilitated by the association of the phosphorylated receptors with the cytoplasmic adaptor, ␤-arrestin (15). The receptor-␤-arrestin complex associates with clathrin and accessory proteins involved in the formation of clathrin-coated pits, ultimately leading to receptor internalization (16,17). While mediating the inhibition of G-protein functions, both G-protein-coupled receptor kinases and ␤-arrestins also activate G-protein-independent signaling events by direct association with a numb...

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

“…␤-Arrestins Localize to the Cell Periphery in D6-expressing HEK293 Cells-GFP-tagged ␤-arrestin-1 is reported to co-localize with D6-RFP throughout transfected rat RBL-2H3 cells (14). We investigated the impact of untagged human wtD6 expression on ␤-arrestin distribution in human HEK293 cells.…”

“…This enables iterative rounds of chemokine internalization through clathrin-coated pits without the need for signaling, with chemokine simply associating with surface D6 molecules destined for internalization (12)(13)(14). Consequently, Ͼ95% of D6 is located in early and recycling endosomal compartments where it shows remarkable stability, presumably by avoiding transit to lysosomes (12).…”

“…Internalization of untagged D6 in HEK293 cells is not inhibited by dominant-negative ␤-arrestin (12). However, red fluorescent protein (RFP)-tagged human D6 (D6-RFP) reportedly colocalizes with green fluorescent protein (GFP)-tagged ␤-arrestin-1 throughout rat basophilic leukemia (RBL)-2H3 cells, and the subcellular distribution of D6-RFP is described as ␤-arrestindependent in mouse embryo fibroblasts (MEFs) (14).…”

Increased inflammation in mice deficient for the chemokine decoy receptor D6

“…Differently from other chemokine receptors [11], D6 expression is restricted to non-hematopoietic cells such as endothelial cells lining afferent lymphatics of skin [12], and placenta [9]. D6 binds most inflammatory, but not homeostatic, CC chemokines [9,13], internalizes constitutively [14,15] and targets the ligand for degradation [16].…”

Tuning inflammation in tuberculosis: the role of decoy receptors

“…The molecular mechanisms of this recycling are still unclear even if it is believed that the D6 receptor is subject to a constitutive ligand-independent phosphorylation [9], helping mediate ligand-independent exocytosis, and that b-arrestins are not necessary for D6 internalizzation. Conversely, other reports suggest that D6 is not phosphorylated and its constitutive internalization is b-arrestins-dependent [10].…”