Purification and biochemical characterization of the D6 chemokine receptor

Abstract

There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1beta (macrophage inflammatory protein-1beta) and by complete binding of the receptor to a MIP-1alpha affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that >80% is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization described here, leads the way towards work aimed at generating the three-dimensional structure of this seven-transmembrane-spanning receptor.

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

“…2A). We previously reported that wtD6 is constitutively phosphorylated in murine L1.2 cells (34). This is also the case in HEK293 cells; anti-D6 immunoprecipitates from 32 P-labeled HEK293 cells only contained a labeled protein of equivalent molecular weight to D6 when the cells expressed wtD6 (Fig.…”

Identification and expression analysis of an atypical chemokine receptor-2 (ACKR2)/CC chemokine binding protein-2 (CCBP2) in rainbow trout (Oncorhynchus mykiss)

“…A potential N-linked glycosylation site is also present in this region in rainbow trout, Atlantic salmon, spotted gar and Mexican tetra at the N-terminus of ACKR2 but is missing in zebrafish. This site is glycosylated in human ACKR2 [49]. Interestingly, the lobe-finned coelacanth ACKR2 has two potential N-linked glycosylation sites in the alignment, one that is conserved in tetrapod ACKR2 molecules and the other conserved in salmonid ACKR2 homologues.…”

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

“…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].…”

“…2009. 39: 342-351 distribution using either GFP tagging or anti-D6 antibodies have indicated that cellular D6 in excess of 95% is present in intracellular vesicles with only low levels of D6 being seen on the cell membrane [55,56]. These intracellular vesicles are early/ recycling endosomes that are central to D6 function.…”