Purification and biochemical characterization of the D6 chemokine receptor

Blackburn, P. E., Simpson, C. V., Nibbs, R. J. B. , O'Hara, M., Booth, R., Poulos, J., Isaacs, N. W. and Graham, G. J. (2004) Purification and biochemical characterization of the D6 chemokine receptor. Biochemical Journal, 379(2), p. 263. (doi:10.1042/BJ20031266)

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Publisher's URL: http://dx.doi.org/10.1042/BJ20031266

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-1b (macrophage inflammatory protein-1b) and by complete binding of the receptor to a MIP-1a 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:O'Hara, Mrs Maureen and Nibbs, Professor Rob
Authors: Blackburn, P. E., Simpson, C. V., Nibbs, R. J. B., O'Hara, M., Booth, R., Poulos, J., Isaacs, N. W., and Graham, G. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Biochemical Journal
Publisher:Portland Press Ltd.
ISSN:0264-6021
ISSN (Online):1470-8728
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