title: 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 beta-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.
authors.affiliation: Division of Immunology, Infection and Inflammation, Glasgow University, Glasgow G12 8TA, Scotland, United Kingdom.
authors.forename: Clare V
authors.initials: CV
authors.lastname: McCulloch
authors.forename: Valerie
authors.initials: V
authors.lastname: Morrow
authors.forename: Sandra
authors.initials: S
authors.lastname: Milasta
authors.forename: Iain
authors.initials: I
authors.lastname: Comerford
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Graham
authors.forename: Neil W
authors.initials: NW
authors.lastname: Isaacs
authors.forename: Robert J B
authors.initials: RJ
authors.lastname: Nibbs
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D054405:Chemokine CCL3 / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D054447:Receptors, CCR10 / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins; D000097628:Chemokine Receptor D6
chemical_list: D019390:Arrestins; D054405:Chemokine CCL3; D054447:Receptors, CCR10; D000071557:beta-Arrestins; D000097628:Chemokine Receptor D6; C515429:CCL3 protein, human