Agonist-induced Endocytosis of CC Chemokine Receptor 5 Is Clathrin Dependent

Abstract

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. INTRODUCTIONChemokine receptors are G protein-coupled receptors (GPCRs) that are activated by chemoattractant cytokines called chemokines. They play key roles in a variety of developmental and chemotactic events (Rossi and Zlotnik, 2000;Horuk, 2001). The CC chemokine receptor 5 (CCR5) is specifically expressed on subsets of leukocytes that are recruited to sites of inflammation by the CC chemokines and CCR5 ligands, CCL3 (macrophage inflammatory protein [MIP]-1␣), CCL4 (MIP-1␤), CCL5 (regulated on activation normal T-cell expressed and secreted [RANTES]), CCL8 (monocyte chemoattractant protein-2), and CCL3L1 (LD78␤). In addition, together with CD4, CCR5 is a major cellular receptor for the human (HIV-1 and HIV-2) and simian immunodeficiency viruses (Simmons et al., 2000).Chemokine receptor agonists are able to inhibit HIV infection of susceptible cells in vitro (Cocchi et al., 1995;Bleul et al., 1996;Oberlin et al., 1996). We and others have established that chemokines trigger endocytosis of cell surface chemokine receptors and that this internalization is a major component of the mechanism of chemokine inhibition of viral infection (Alkhatib et al., 1997;Amara et al., 1997;Signoret et al., 1997;Mack et al., 1998;. Agonist binding induces rapid CCR5 internalization and accumulation of the internalized receptor in recycling endosomes (Mack et al., 1998;Signoret et al., 1998. Although this internalization has been studied superficially, little is known of how these receptors are recruited into endocytic organelles. Initial studies of immunolabeled cryosections from agonist-treated Chinese hamster ova...

Chemokine receptor internalization and intracellular trafficking

“…However, in some cell types, one pathway may play a more dominant role over the other in mediating internalization of the receptor. For example, in chinese hampster ovary (CHO) and mink lung endothelial cells, CCR5 internalizes mainly through the clathrin-dependent pathway [5], while in primary T cells or HEK293 cells, CCR5 predominantly internalizes through caveoli [3]. The result is that receptors undergo a much faster receptor internalization in CHO and mink lung endothelial cells than those in primary T cells and HEK293 cells [3,5].…”

“…Venkatesan et al report that CCR5 is internalized to vesicles that partially colocalized with endogenous caveolin [3], but Signoret et al conclude that CCR5 endocytosis was completely independent of caveolae [5]. The mechanism by which CCR5 internalizes is controversial and needs to be investigated further.…”

CCR5 expression on monocytes and T cells: Modulation by transmigration across the blood–brain barrier in vitro

“…These adaptive mechanisms (active in many G-protein coupled receptors [GPCRs]) operate within different time frames after ligand-receptor interactions [8][9][10][11]. Processes underlying GPCR modulation include ligand-induced internalization, with subsequent recycling or degradation.…”

“…β-arrestins also serve as adaptor proteins that link phosphorylated CCR5 to clathrin in order to mediate receptor internalization via endocytosis [8,9,11,13]. Arrestins can additionally act as adaptors in signaling processes [14].…”

Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5

“…18 Receptor endocytosis is thought to regulate homologous desensitization or the decrease in cell migration observed at higher ligand concentrations. 42,43 Therefore, we measured the kinetics of internalization for these CCR5 variants, when they were exposed to CCL5. It has been previously reported that exposure to ligand for 60 min allows for the maximum internalization of CCR5, 42 all tested variants showed similar internalization at this time point.…”