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.
Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically-applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of antibodies in this context and alternative approaches are required. Here we demonstrate that appropriately labelled chemokines are at least as effective in detecting their cognate receptors as commercially available antibodies. We also demonstrate the utility of biotinylated-chemokines as cell-sorting reagents. Specifically we demonstrate, in the context of CCR7 (essential for lymph node-homing of leukocytes), the ability of biotinylated-CCL19 with magnetic-bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability and the sorting is effective for both T cells and dendritic cells. Importantly the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile and applicable to numerous cell-therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications.
PAGE 7977:The original version of Fig. 4 contained an error. The same image was used in the "wtD6" and "D6-Ala6" panels. We have now replaced the "D6-Ala6" panel with the correct image. This revision does not change the results or the interpretation of the data.
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