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Vol. 16, Issue 2, 902-917, February 2005
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* Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom;
Department of Immunology, Georg-August-University, 37075 Göttingen, Germany
Submitted August 10, 2004;
Revised October 19, 2004;
Accepted December 1, 2004
Monitoring Editor: Jean Gruenberg
The signaling activity of several chemokine receptors, including CC chemokine receptor 5 (CCR5), is in part controlled by their internalization, recycling, and/or degradation. For CCR5, agonists such as the chemokine CCL5 induce internalization into early endosomes containing the transferrin receptor, a marker for clathrin-dependent endocytosis, but it has been suggested that CCR5 may also follow clathrin-independent routes of internalization. Here, we present a detailed analysis of the role of clathrin in chemokine-induced CCR5 internalization. Using CCR5-transfected cell lines, immunofluorescence, and electron microscopy, we demonstrate that CCL5 causes the rapid redistribution of scattered cell surface CCR5 into large clusters that are associated with flat clathrin lattices. Invaginated clathrin-coated pits could be seen at the edge of these lattices and, in CCL5-treated cells, these pits contain CCR5. Receptors internalized via clathrin-coated vesicles follow the clathrin-mediated endocytic pathway, and depletion of clathrin with small interfering RNAs inhibits CCL5-induced CCR5 internalization. We found no evidence for CCR5 association with caveolae during agonist-induced internalization. However, sequestration of cholesterol with filipin interferes with agonist binding to CCR5, suggesting that cholesterol and/or lipid raft domains play some role in the events required for CCR5 activation before internalization.
Abbreviations used: BM, binding medium; CCP, clathrin-coated pit; CCR5, CC chemokine receptor 5; CCV, clathrin-coated vesicle; CHC, clathrin heavy chain; CHO, Chinese hamster ovary; CLC, clathrin light chain; CTxB, cholera toxin B subunit; EM, electron microscopy; GPCR, G protein-coupled receptor; HTf-R, human transferrin receptor; Mv-1-Lu, mink lung endothelial cells; PAG, protein A-gold; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RBL, rat basophilic leukemia; Tf, transferrin; siRNA, small interfering RNA.
Corresponding author. E-mail address: m.marsh{at}ucl.ac.uk.
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