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- Materials and Methods
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 NH4Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the β2-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1β (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-1β dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.
Chemokine receptors are members of the seven transmembrane domain (7TM), heterotrimeric G-protein coupled receptor (GPCR) superfamily. They function primarily in immune and inflammatory responses, but have also been shown to play roles in development, angiogenesis and haematopoiesis (1,2). The CC (β)-chemokine receptor 5 (CCR5) is also an essential cofactor for the entry of R5 tropic strains of the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) (3). Cells susceptible to HIV can be protected by treatment with CCR5 agonists, including the CC-chemokines RANTES (regulated on activation normal T-cell expressed and secreted; CCL5), macrophage inflammatory proteins (MIP)1α (CCL3) and 1β (CCL4). Agonist-induced chemokine receptor endocytosis is the major mechanism through which this protection occurs (4–6).
Endocytosis regulates the cell surface expression of many GPCRs and has been implicated in desensitisation, resensitisation and down-modulation of these receptors (7). We previously demonstrated that agonists trigger CCR5 internalisation in Clathrin coated vesicles (CCVs), leading to an accumulation of internalised receptors in perinuclear recycling endosomes (RE) (8). When the agonist was removed, the cell surface pool of CCR5 was recovered by recycling of the internalised receptors in a ligand-free and resensitised form (8,9). However, experiments with the N-terminally modified aminooxypentane (AOP)-RANTES suggested that agonist–receptor dissociation was not required for recycling. CCR5 molecules on which the agonist binding site(s) remain occupied could recycle, but on reaching the plasma membrane these molecules were rapidly re-internalised and returned to the RE (8). Thus agonist dissociation may not be essential for CCR5 recycling, and the endosomal recycling machinery may not distinguish between agonist-occupied and unoccupied receptors.
One of the key regulatory mechanisms used by endocytic organelles is acidification. For many receptor-ligand complexes internalised by clathrin-mediated endocytosis, exposure to low pH in endosomes facilitates ligand-receptor dissociation and the subsequent trafficking of receptors and/or ligands (10). Indeed studies with the β2-adrenergic receptor (β2-AR) have suggested that low pH-induced agonist dissociation is required for receptor resensitisation and recycling, through processes that involve recruitment of a GPCR phosphatase (GRP) and dephosphorylation of the activated receptor (7). For the β2-AR at least, these events are believed to occur in early sorting endosomes (SE), though little detail exists. Our studies suggest that following endocytosis, CCR5 enters the SE and is rapidly transferred to RE (8). Currently nothing is known about how these sorting events occur or whether transit to the RE is functionally significant for CCR5 activity and cycling.
In this study we investigated whether acidification of endosomes is required for CCR5 recycling. To this end, we analysed the fate of internalised CCR5 and two CC chemokine agonists in cells treated with agents that increase endosomal pH, specifically bafilomycin A, monensin and NH4Cl. We found that, unlike the β2-AR, CCR5 recycling is not regulated by endosomal acidification. Furthermore, RANTES and MIP-1β cannot be eluted from CCR5 at physiological pH, suggesting that ligand dissociation is a pH-independent event for this receptor. Nevertheless, low endocytic or lysosomal pH does appear to be required for efficient degradation of CCR5 ligands removed from internalised receptors.
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- Materials and Methods
It has long been established that sorting of plasma membrane receptor-ligand complexes internalised by receptor-mediated endocytosis through CCVs occurs in the tubulo-vesicular compartments that make up endosomes. Frequently, ligands are removed from the receptors and degraded, while the receptors recycle to the cell surface. Alternatively, receptors themselves, with or without ligands, may be targeted to lysosomes and degraded or delivered to other cellular locations. A major factor that contributes to the control of these sorting events is the acidification of endocytic compartments. This acidification has been shown to be important in facilitating the dissociation of some ligands from their cognate receptors and, in some cases, for receptors to couple to sorting machineries (10,11,23). Here we show that for CCR5, exposure to acid pH is not required for chemokine dissociation or receptor recycling.
Many GPCRs undergo endocytosis after activation by specific agonists. Internalisation has been linked in some cases to receptor down-regulation, by sorting to lysosomes and degradation. In other cases internalisation is associated with resensitisation, whereby activated receptors can be returned to the cell surface in a non-activated or resensitised form. One paradigm for resensitisation is seen with β2-AR. Agonist binding to this receptor results in phosphorylation of the carboxyl-terminal domain of the receptor by a GPCR kinase (GRK) and subsequent arrestin-dependent internalisation in CCVs (7). These receptors may then be returned to the cell surface in a resensitised form and are again able to respond to agonist. Based on results from several laboratories, a view has emerged that vesicular acidification regulates the recycling of at least some internalised GPCRs to the cell surface (22,24,25). Following endocytosis and delivery to endosomes, the GPCR-agonist complexes are exposed to mildly acidic pH that induces dissociation of the agonist from the receptor. This may involve conformational changes in the receptor that, in the case of β2-AR, render the cytoplasmic carboxyl-terminal domain accessible to a GPCR phosphatase that dephosphorylates the activated receptor (20). The resensitised receptors then return to the cell surface through mechanisms that remain to be understood but may require specific recycling proteins (26).
Two distinct recycling pathways from SE have been described. First, a pathway in which receptors are returned directly to the cell surface, as appears to be the case for β2-AR. Alternatively, a route involving RE has been observed for a number of receptors including the transferrin receptor. We have previously found that CCR5, a GPCR for several inflammatory CC chemokines, undergoes efficient agonist-induced endocytosis and recycling through RE (8). In addition, we observed with AOP-RANTES that although resensitised receptors could recycle to the cell surface, resensitisation might not be essential for recycling (8). The main indication for this was the observation that after treatment with AOP-RANTES, CCR5 molecules with occupied ligand binding sites could recycle, and that on reaching the cell surface these CCR5 molecules would re-engage the endocytic machinery and be re-internalised. This suggested that for CCR5 the endosomal machinery is not able to distinguish ligand-bound from empty molecules, and that dephosphorylation may not be required for transport of receptors from endosomes to the cell surface. Here we investigated whether acidification of endosomes is required for CCR5 recycling.
We used three distinct compounds to raise the pH of endocytic organelles: bafilomycin A, an inhibitor of the vacuolar proton pump; monensin, a carboxylic ionophore; and the weak base NH4Cl (14,27,28). None of these drugs affected the kinetics of ligand-induced CCR5 internalisation, nor the intracellular trafficking of internalised CCR5 through SE to RE. Significantly, the subsequent recycling of CCR5 from RE to the plasma membrane was only marginally affected by endosomal alkalinisation. Similar recycling kinetics were measured in the presence and absence of the three agents. In addition, no difference was seen if MIP-1β was used instead of RANTES, or when assays were performed in CHO or CEM cells, indicating that these properties of CCR5 are independent of the agonist used, or the cell type in which the receptor is expressed. Some modest inhibition of recycling was seen in the presence of bafilomycin A or another V-ATPase inhibitor, concanamycin A (data not shown), which may be due to effects of V-ATPase inhibition additional to that of endosomal alkalinisation (13,29,30).
In many cases, the low pH environment in endosomes promotes dissociation of internalised ligand-receptor complexes, allowing ligand-free receptor to recycle to the cell surface (7,10,11). The finding that CCR5 can recycle in cells treated with bafilomycin A, monensin and NH4Cl raises the question of how ligand is removed from this receptor. We assessed the effect of acidic media on chemokine-CCR5 complexes directly by measuring elution of cell surface CCR5-bound chemokines. Radiolabelled RANTES or MIP-1β were only released from plasma membrane receptors when exposed to acid solutions with a pH < 4.0. This pH range is substantially lower than that normally found in the endocytic pathway, suggesting that chemokine dissociation from internalised CCR5 is not enhanced by exposure to low pH.
In previous studies with AOP-RANTES, and in the experiments reported here with RANTES, we find that recycled receptors can re-internalise into the endocytic pathway. For AOP-RANTES treated cells, at least, recycling receptors appear to remain ligand-occupied. We assume that those receptors re-internalised on RANTES treated cells have retained RANTES. As shown here, we also find that CCR5 remains phosphorylated for a prolonged period after a short treatment with agonist, perhaps supporting the view that RANTES dissociation, at least, is inefficient and that agonist-occupied receptors are not dephosphorylated during recycling. Alternatively, if they are dephosphorylated, they may be rapidly re-phosphorylated. By contrast, in the presence of TAK-779 agonist dissociation is promoted (not shown), receptor re-internalisation is inhibited (Figures 3 and 4), and the receptor is efficiently dephosphorylated (Figure 5). Significantly, exposure to low pH is not required for dephosphorylation (Figures 5B,C). Thus, agonist dissociation would be primarily responsible for marking receptors for dephosphorylation. When this step is pH-independent, dephosphorylation is also pH-independent. Where exactly dephosphorylation of CCR5 occurs is unclear. All receptors may be dephosphorylated in endosomes, but the agonist-occupied receptors rapidly rephosphorylated. Alternatively, receptors may recycle in a phosphorylated form, and following TAK-779-induced RANTES dissociation, undergo dephosphorylation at the plasma membrane. More detailed analyses with CCR5 specific anti-phosphopeptide antibodies might distinguish between these possibilities.
The observation that ligand-occupied CCR5 can recycle suggests that chemokine-occupied receptors may undergo multiple rounds of transit through the endosomal system. Whether there is a mechanism for releasing ligand from CCR5 remains unclear. Nevertheless, degraded TCA-soluble, 125I-RANTES-derived activity can be recovered from the medium of RANTES treated cells. Thus some RANTES is released from internalised receptors and this material is degraded by acid hydrolases, or requires an acidification-dependent transport step for delivery to the degradative compartment.
A role for acidification in GPCR recycling was suggested by the finding that monensin inhibited the return of internalised β2-AR to the plasma membrane (22). The neurokinin 1 (NK1) receptor and the cannabinoid (CB1) receptor have also been shown to require vesicular acidification for recycling (24,25). Here, with CCR5, we show that recycling is pH-independent. Overall our studies indicate that endosomal acidification is not a general regulator of GPCR recycling. As it emerges that distinct intracellular itineraries exist for different GPCRs, specific mechanisms may also exist to control GPCR sorting and recycling. In the case of CCR5 these mechanisms remain to be identified.