title: Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells.
abstract: OBJECTIVE
  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-1alpha [CCL3], MIP-1beta [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.
  
  
  METHODS
  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.
  
  
  RESULTS
  MIP-1alpha [CCL3], MIP-1beta [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-1alpha-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
  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.
authors.affiliation: Institute of Cell Signalling, Medical School, Queens Medical Centre, Nottingham, UK.
authors.forename: J
authors.initials: J
authors.lastname: Longden
authors.forename: E-L
authors.initials: EL
authors.lastname: Cooke
authors.forename: S J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000577:Amides / Q000494:pharmacology; D000626:Aminopeptidases / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D065100:CCR5 Receptor Antagonists; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D054405:Chemokine CCL3 / Q000494:pharmacology; D054407:Chemokine CCL4 / Q000494:pharmacology; D018946:Chemokine CCL5 / Q000378:metabolism / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects*; D049452:Green Fluorescent Proteins; D006801:Humans; D049408:Luminescent Agents; D018613:Microscopy, Confocal; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000819:agonists / Q000378:metabolism; D013997:Time Factors
chemical_list: D000577:Amides; D065100:CCR5 Receptor Antagonists; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D049408:Luminescent Agents; D000644:Quaternary Ammonium Compounds; C108335:RANTES, Met-; D019713:Receptors, CCR5; D049452:Green Fluorescent Proteins; C119369:TAK 779; D000626:Aminopeptidases; D002118:Calcium