title: pH-independent endocytic cycling of the chemokine receptor CCR5.
abstract: Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.
authors.affiliation: Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK. n.signoret@ucl.ac.uk
authors.forename: Nathalie
authors.initials: N
authors.lastname: Signoret
authors.forename: Thierry
authors.initials: T
authors.lastname: Christophe
authors.forename: Martin
authors.initials: M
authors.lastname: Oppermann
authors.forename: Mark
authors.initials: M
authors.lastname: Marsh
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D018946:Chemokine CCL5 / Q000494:pharmacology; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008024:Ligands; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D019713:Receptors, CCR5 / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled; D013997:Time Factors
chemical_list: D018946:Chemokine CCL5; D008024:Ligands; D018942:Macrolides; D018343:Receptors, Adrenergic, beta-2; D019713:Receptors, CCR5; D043562:Receptors, G-Protein-Coupled; C057620:bafilomycin A; D049452:Green Fluorescent Proteins; D008985:Monensin