Research Paper
Subject Category: Molecular and cellular mechanisms
British Journal of Pharmacology (2008) 153, 1513–1527; doi:10.1038/sj.bjp.0707691; published online 28 January 2008
Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells
J Longden1,2,3, E-L Cooke2,4 and S J Hill1
- 1Institute of Cell Signalling, Medical School, Queens Medical Centre, Nottingham, UK
- 2Advanced Science and Technology Laboratory, AstraZeneca Research and Development Charnwood, Loughborough, Leicestershire, UK
Correspondence: Professor SJ Hill, Institute of Cell Signalling, Medical School, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK. E-mail: stephen.hill@nottingham.ac.uk
3Current address: Eskitis Institute of Cell and Molecular Therapies, Griffith University, Nathan, Queensland 4111, Australia.
4Current address: Cancer BioScience, AstraZeneca Research and Development Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
Received 12 September 2007; Revised 6 November 2007; Accepted 4 December 2007; Published online 28 January 2008.
Abstract
Background and purpose:
The CCR5 chemokine receptor is a member of the G protein-coupled receptor (GPCR) family that is expressed by macrophages, memory T-lymphocytes and dendritic cells and is activated by chemotactic proteins (e.g. MIP-1
[CCL3], MIP-1
[CCL4] and RANTES [CCL5]). CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus-1 (HIV-1) and some chemokines can inhibit HIV-1 infection by stimulating CCR5 receptor endocytosis. The aim of this study was to evaluate the effect of CCR5 antagonists on CCR5 endocytosis.
Experimental approach:
The effects of CCR5 agonists and antagonists on receptor internalization in CHO cells, expressing a C-terminal green fluorescent protein-tagged human CCR5 receptor (CCR5-GFP), were quantified using a confocal imaging plate reader.
Key results:
MIP-1 [CCL3], MIP-1 [CCL4] and RANTES [CCL5] were all able to stimulate potently the internalization of CCR5-GFP. This effect was inhibited by the non-peptide antagonist TAK 779. The CCR5 peptide antagonist met-RANTES antagonized MIP-1-mediated increases in intracellular free calcium but was also able to stimulate a substantial internalization of the human CCR5-GFP receptor. However, CHO cells exhibited an aminopeptidase activity that was able to metabolize sufficient met-RANTES into an agonist metabolite capable of stimulating calcium mobilization via CCR5 receptors in naïve cells.
Conclusions and implications:
These data suggest that there is an endogenous aminopeptidase activity on the surface of CHO cells, that produces a slow internalization of the receptor following a time-dependent conversion of receptor-bound met-RANTES from a CCR5 receptor antagonist into a CCR5 agonist molecule.
Keywords:
receptor internalization, CCR5 receptor, met-RANTES, G-protein-coupled receptors, antagonist stimulated, endosomes, aminopeptidase
Abbreviations:
CCL, official chemokine ligand nomenclature name; CHO, Chinese hamster ovary; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HIV-1, human immunodeficiency virus, type 1; MAP kinase, mitogen-activated protein kinase; MIP, macrophage inflammatory protein; PBS, phosphate-buffered saline; RANTES, released on activation normal T cell expressed and secreted
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