Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent.
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.
Signoret
Nathalie
N
Cell Biology Unit, Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.
Hewlett
Lindsay
L
Wavre
Silène
S
Pelchen-Matthews
Annegret
A
Oppermann
Martin
M
Marsh
Mark
M
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D054407:Chemokine CCL4; D019742:Chemokines, CC / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects*; D042783:Endothelial Cells / Q000648:ultrastructure; D004847:Epithelial Cells / Q000648:ultrastructure; D005372:Filipin / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006834:Hydrazines; D008168:Lung / Q000166:cytology; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008907:Mink; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D019713:Receptors, CCR5 / Q000819:agonists* / Q000378:metabolism* / Q000648:ultrastructure
C417561:Alexa 488 hydrazide; D000900:Anti-Bacterial Agents; C496535:Ccl4 protein, mouse; D054407:Chemokine CCL4; D019742:Chemokines, CC; D002966:Clathrin; D005456:Fluorescent Dyes; D006834:Hydrazines; D019402:Macrophage Inflammatory Proteins; D034741:RNA, Small Interfering; D019713:Receptors, CCR5; D049452:Green Fluorescent Proteins; D005372:Filipin