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Keywords:

  • CCR5;
  • chemokine;
  • chemokine receptor;
  • endocytosis;
  • recycling

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

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.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

We have previously shown that in CCR5-transfected CHO cells, RANTES and AOP-RANTES induce CCR5 endocytosis and sorting to RE. Immunofluorescence and electron microscopic analyses have indicated that CCR5 internalises through CCVs and rapidly transits through early peripheral SE before reaching RE (8). For many receptors, endosome acidification is essential for the sorting events occurring during endocytosis (11). In order to assess whether this acidification influences CCR5 trafficking and recycling, we compared the fate of agonist-activated CCR5 receptors in the presence or absence of reagents that alkalinise the endocytic pathway. The immediate effect of these compounds is to raise the lumenal pH of endocytic organelles, but additional compound-specific effects can also occur (11–13). Here, we used three distinct reagents to alter the intravesicular pH: the vacuolar (H+)-ATPase (V-ATPase) inhibitor bafilomycin A, the carboxylic ionophore monensin, and the weak base NH4Cl.

Alkalinisation of endocytic organelles

We verified that bafilomycin A, monensin and NH4Cl were able to alter the acidic environment of endosomes in CHO-CCR5 cells by measuring their capacity to prevent infection by Semliki Forest virus (SFV). SFV infection involves endocytosis and penetration of the nucleocapsid to the cytosol by pH-dependent fusion of the viral and endosomal membranes. It has previously been shown that weak bases and ionophores block SFV infection by raising endosomal pH (14,15). A recombinant SFV-GFP virus was initially titrated on CHO-CCR5 cells treated or not with 10 μm monensin (16). Infected cells were detected 6 h post infection by the presence of GFP in the cytoplasm (Figure 1A). The majority of the cells were infected when exposed to 2 × 105 infectious units (IU) of SFV-GFP. By contrast, only a few infected cells were visible even at a dose of 4 × 106 IU in the presence of monensin. This initial experiment indicated that monensin could efficiently inhibit SFV infection. From this experiment, we determined 4 × 105 IU as an optimal dose for infection. We then compared the infectivity of CHO-CCR5 cells when treated with 10 nm bafilomycin A, 10 μm monensin or 20 mm NH4Cl, concentrations previously used to increase the pH in endosomes and lysosomes (13,15,17). The proportion of infected cells was determined for each condition by counting the number of GFP-expressing cells. All three reagents strongly inhibited SFV-GFP entry, with monensin being the most efficient and bafilomycin A the least potent of the three. Thus bafilomycin, monensin and NH4Cl can rapidly increase the pH of endocytic organelles in CHO cells.

image

Figure 1. Inhibition of SFV infection.A) SFV-GFP was tit-rated on CHO-CCR5 cells grown on coverslips. Following 5 min pretreatment in BM alone or BM containing monensin, cells were infected with different doses of SFV-GFP (IU/well) in the absence or presence of monensin. SFV infected cells were visible 6 h post infection as GFP accumul-ated in their cytoplasm. Samples were analysed with a Zeiss Axios-kop microscope and digital images collected using a Hamamatsu Orca Camera. Bar = 20 μm. B) CHO-CCR5 cells grown on coverslips were pretreated and infected with SFV-GFP (4 × 105 IU/well) in BM alone or BM containing bafilomycin A, monensin or NH4Cl. At 6 h post infection the number of infected cells was determined by counting GFP expressing cells. The results are expressed as the percent of infected cells present in each culture (300 cells counted per sample). The graph shows the mean ± s.d. of a representative experiment performed in duplicate.

Endosomal alkalinisation does not affect the distribution of internalised CCR5

To determine whether endosomal acidification is important for the internalisation of CCR5, we investigated the distribution of agonist-treated CCR5 in the presence of bafilomycin A, monensin or NH4Cl. CHO-CCR5 cells were treated for 90 min at 37 °C with 125 nm RANTES in the presence or absence of the drugs and CCR5 localisation was then analysed by immunofluorescence using the anti-CCR5 mAb MC-5. As we previously reported, internalised CCR5 is delivered to endosomal vesicles located both in the periphery of the cell and close to the nucleus (Figure 2A). The perinuclear vesicles correspond to RE and the peripheral vesicles are components of SE, as indicated by co-labelling with antibodies against EEA1 (data not shown). Very similar distributions were seen for CCR5 internalised in the presence of bafilomycin A, monensin 2 and NH4Cl (Figure 2). This indicates that the internalisation of CCR5, and its intracellular sorting and transport to RE, are not dependent on endosomal acidification.

image

Figure 2. Immunofluorescence localisation of CCR5. CHO-CCR5 cells grown on coverslips were incubated for 90 min at 37 °C with 125 nm RANTES in the absence (A) or presence of bafilomycin A (B), monensin (C), or NH4Cl (D). CCR5 was then detected on fixed and permeabilised cells using 488MC-5, and the samples were analysed by confocal microscopy. The figure shows micrographs of single confocal sections approxi-mately 0.5 μm thick. Bar = 20 μm.

CCR5 recycling in the presence of TAK-779

Following removal of the ligand from RANTES-treated CHO-CCR5 cells, internalised CCR5 is recycled to the cell surface (8). A fraction of these recycled receptors do not remain at the plasma membrane but are returned to RE. To measure CCR5 molecules undergoing a single round of recycling to the cell surface, we used a specific CCR5 antagonist to trap all recycling receptors at the cell surface and prevent re-endocytosis of recycled CCR5. TAK-779, a synthetic anilide derivative with a quaternary ammonium moiety, has been shown to inhibit the binding of CC chemokines, including RANTES and MIP-1β, to CCR5 (18,19). We found that TAK-779 displaced RANTES prebound to CCR5 at 4 °C and prevented RANTES-induced CCR5 internalisation (data not shown). We tested morphologically (Figure 3A) and biochemically (Figure 3B) whether TAK-779 could retain recycled CCR5 molecules at the plasma membrane. For immunofluorescence, CHO-CCR5 cells were exposed to RANTES for 30 min at 37 °C to induce CCR5 internalisation. The RANTES-containing medium was then removed, replaced with medium containing TAK-779 and the cells then incubated for 60 min at 37 °C to allow CCR5 to recycle. CCR5 distribution was analysed on fixed and permeabilised cells stained with 488MC-5. On untreated cells (Figure 3A, panel a) CCR5 was seen to be located primarily at the cell surface. After 30 min of treatment with RANTES, most of the cell surface staining had disappeared and the CCR5 molecules were seen in intracellular vesicles, most of which were located close to the nucleus (Figure 3A, panel b). After reincubation for 60 min in the presence of TAK-779, prominent cell surface staining was again observed with very little punctate intracellular staining remaining (Figure 3A, panel c). In contrast to our previous recycling experiments in the absence of TAK-779, when significant intracellular staining was observed due to re-endocytosis of recycled receptors (8), inclusion of TAK-779 in the medium caused recycled CCR5 to be retained at the cell surface.

image

Figure 3. Analysis of CCR5 recycling on RANTES-treated cells.A) CHO-CCR5 cells grown on coverslips were incubated in BM alone (a) or with RANTES for 30 min at 37 °C to induce receptor internalisation (b). The ligand was then washed away and the cells were incubated with TAK-779 for a further 1 h at 37 °C to allow CCR5 recycling (c). Parallel samples were treated as in panel c but in the continuous presence of bafilomycin A (d), monensin (e), or NH4Cl (f). CCR5 was then detected by staining with 488MC-5 on fixed and permeabilised cells. Samples were analysed and recorded as described in Figure 1. Scale bar = 20 μm. B, C) CHO-CCR5 cells (B) or CEMss-CCR5 cells (C) were treated in suspension for up to 60 min at 37 °C with RANTES. Following several washes in 4 °C BM (arrow), the cells were incubated in medium containing TAK-779 for up to 60 min at 37 °C. The whole experiment was performed either in BM alone (□) or in BM containing bafilomycin A (◆), monensin (•) or NH4Cl (▴). Cell surface CCR5 down-modulation and recycling were monitored by immunofluorescent staining with 488MC-5 for CHO-CCR5 cells, or MC-5 and a FITC-conjugated goat anti-mouse Ab for CEMss-CCR5 cells. Samples were analysed by flow cytometry and the results are expressed as the percent of the fluorescence on untreated cells. The graph shows the mean ± s.d. of four (B) and three(C) independent experiments.

To analyse recycling quantitatively, CHO-CCR5 cells in suspension were incubated at 37 °C with RANTES for up to 60 min. Ligand-induced CCR5 internalisation was stopped by cooling and washing the cells on ice. An excess of TAK-779 was then added to the suspension and the cells incubated at 37 °C for up to 60 min to allow recycling. Plasma membrane CCR5 receptors were then detected by staining intact cells with 488MC-5 and FACS analysis. RANTES treatment induced a progressive loss of cell surface CCR5 immunoreactivity, reflecting receptor endocytosis, with a maximum reduction of 75% after 30 min incubation (Figure 3B). Immediately after the beginning of the recycling phase, in the presence of TAK-779, we detected an increase in cell surface staining with nearly 100% recovery after a 60-min period. Together, these experiments indicate that in the presence of TAK-779 virtually all internalised CCR5 recycles to, and is retained at, the cell surface.

CCR5 recycling is not inhibited by alkalinisation of endocytic organelles

To examine the influence of endosome acidification on CCR5 recycling, we performed the immunofluorescence and FACS experiments described above on CHO-CCR5 cells in the continuous presence of bafilomycin A, monensin or NH4Cl (Figure 3). As for the cells incubated in the absence of the reagents, RANTES induced CCR5 internalisation and transport to RE (not shown) (8). When cells were then washed and incubated with TAK-779 for 60 min, CCR5 staining was seen mainly at the plasma membrane (Figure 3A, panels d–f), although a few intracellular punctate structures were seen in cells treated with bafilomycin A (Figure 3A, panel d). In cells treated with monensin or NH4Cl virtually all CCR5 appeared to be returned to the cell surface, as seen with control cells (Figure 3A, panels c, e and f). To establish whether these reagents affected the rate and/or extent of cycling, internalisation and recycling were assessed by FACS analysis. Bafilomycin A, monensin or NH4Cl had no detectable effect on the decrease in CCR5 immunoreactivity induced by RANTES treatment, indicating that the reagents did not interfere with RANTES-induced CCR5 endocytosis (Figure 3B). Following removal of RANTES and addition of TAK-779, cell surface CCR5 was observed to recover, as indicated by MC-5 labelling. The extent of recovery varied slightly for the three agents, with 85%, 82% and 69% of the initial cell surface levels recovered after 60 min for NH4Cl, monensin and bafilomycin A, respectively, compared to 92% for the controls (Figure 3B). The slight reduction in the amount of CCR5 recycled to the cell surface in the presence of the three agents is most likely due to long-term effects of endosomal alkalinisation on trafficking through the endocytic pathway.

We estimated the rate of CCR5 recycling to the plasma membrane in the first 10 min of incubation with TAK-779. In control cells, cell surface CCR5 recovered at a rate of 2.9% of the initial cell surface activity per min (Table 1). Very similar rates were seen with monensin (2.7%/min) and NH4Cl (2.7%/min) treated cells. Bafilomycin slowed the rate of recycling to 1.8% per min, but nevertheless significant recycling of CCR5 was observed both in the morphological experiments and in the FACS based assays.

Table 1. Rates for recycling of internalised CCR5 to the plasma membrane of CHO cells (% of initial cell surface labelling/min)
 RANTES mean ± s.d. (n = 4)MIP-1β mean ± s.d. (n = 3)
  1. Recycling rates were determined from the FACS experiments described in Figures 3 and 4 by calculating the percentage of fluorescent signal recovered in the first 10 min following ligand removal.

Medium2.95 ± 0.412.44 ± 0.55
Bafilomycin A1.83 ± 0.450.99 ± 0.20
Monensin2.67 ± 0.521.51 ± 0.12
NH4Cl2.70 ± 0.712.30 ± 0.89

To verify that our results were not cell-type dependent, we repeated the FACS experiment with a T-cell line stably transfected with CCR5 (CEMss-CCR5). The amount of CCR5 on these cells was relatively low compared with the CHO-CCR5 cells. Hence cells were labelled with MC-5 and a secondary fluorescent anti-mouse antibody for analysis. As indicated in Figure 3C, CCR5 recycling was again observed in the presence of bafilomycin A, monensin and NH4Cl. Together, these data indicate that RANTES-induced endocytosis and recycling is not significantly affected by alkalinisation of endocytic compartments.

pH-independent CCR5 recycling is not ligand specific

In addition to RANTES, MIP-1α and MIP-1β are agonists for CCR5. To investigate whether the pH-independent CCR5 recycling was ligand specific, we analysed CHO-CCR5 cells treated with 125 nm MIP-1β by FACS (Figure 4). An identical protocol to that described above for RANTES was used. Cells treated with MIP-1β in binding medium (BM) showed a rapid down-modulation of CCR5 with cell surface CCR5 levels decreased by 60% after 30 min. An increase in cell surface staining was detected soon after removal of the chemokine and addition of TAK-779, with 100% recovery after 60 min. Thus, MIP-1β induced similar internalisation and recycling of CCR5 to that seen with RANTES. CCR5 recycling to the cell surface was also seen in MIP-1β treated cells in the presence of bafilomycin A, monensin or NH4Cl (Figure 4). We noticed that recycling was less efficient in the presence of bafilomycin A and monensin (64% and 73% after 60 min, respectively), but no significant difference was seen in the presence of NH4Cl (Figure 4). The recycling rates calculated for the first 10 min of incubation with TAK-779 were also somewhat reduced with bafilomycin A and monensin (Table 1). Nevertheless, these experiments again indicate that efficient recycling of CCR5 occurs after MIP-1β treatment in the presence of the three different reagents and that pH-independent recycling is not unique to the ligand RANTES.

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Figure 4. MIP-1β-induced endocytosis and recycling of CCR5 on CHO cells. CHO-CCR5 cells were treated with MIP-1β to allow CCR5 down-modulation, and then incubated at 37 °C in the presence of TAK-779 (as in Figure 3). The experiment was performed in the absence (□) or presence of bafilomycin A (◆), monensin (•) or NH4Cl (▴). Cell surface receptors were detected with 488MC-5 and samples analysed by FACS. The graph shows the mean ± s.d. of three independent experiments.

CCR5 dephosphorylation

Dephosphorylation is required for resensitisation of activated GPCRs (7). For the β2-AR, dephosphorylation has been linked to low pH-induced dissociation of agonist, following internalisation into endosomes, and recruitment of a GRP (20). Thus, dephosphorylation and resensitisation are dependent on receptor exposure to low pH. To examine whether dephosphorylation of CCR5 is similarly pH-dependent we first examined agonist-induced phosphorylation in the presence or absence of TAK-779. CHO-CCR5 cells were metabolically labelled with [32P]-orthophosphate, treated with RANTES for 10 min, washed and allowed to recover in agonist-free medium. Analysis of CCR5 by immunoprecipitation, SDS-PAGE and autoradiography showed that in these conditions RANTES treatment induced a rapid and sustained phosphorylation of CCR5 for up to 4 h (Figure 5A, top panel). However, following agonist washout and addition of TAK-779, the initial rapid phosphorylation was followed by complete CCR5 dephosphorylation within 90 min (Figure 5A, lower panel). We also assessed CCR5 phosphorylation and dephosphorylation by immunofluorescence using an antibody specific for phosphorylated CCR5. The M5/4 monoclonal antibody recognises a phosphopeptide including serine 349, a GRK-specific phosphorylation site in the CCR5 cytoplasmic domain, as described for another monoclonal antibody E11/19 (21). CHO-CCR5 cells on glass coverslips were treated with RANTES for 10 min before being washed and re-incubated in medium containing TAK-779 for up to 90 min. CCR5 phosphorylation was analysed on fixed and permeabilised cells co-stained with M5/4 and MC-5 (Figure 5B, left panels). In untreated cells, the pool of CCR5 molecules was visualised at the cell surface by MC5 staining, and no labelling for M5/4 was detected. After 10 min of agonist treatment, M5/4 labelling was seen both at the plasma membrane and on intracellular vesicles, consistent with CCR5 being phosphorylated and internalised. During the chase with TAK-779, the internal MC5 signal weakened and the original cell surface pattern was recovered after 90 min due to CCR5 recycling. In parallel, the fluorescent signal of M5/4 decreased with time and no staining for phosphorylated CCR5 was visible after 90 min. Thus, in the presence of TAK-779, CCR5 is dephosphorylated during its recycling to the plasma membrane.

image

Figure 5. RANTES-induced CCR5 phosphorylation.A) CHO-CCR5 cells were labelled with [32P]-orthophosphate and incubated in medium with RANTES for up to 10 min, ligand was washed away and the cells re-incubated in medium alone for up to 4 h (top panel), or in medium containing TAK-779 for up to 90 min (lower panel). CCR5 molecules were immunoprecipitated, separated by SDS-PAGE and the gels exposed for autoradiography. B) CHO-CCR5 cells on coverslips were preincubated for 30 min at 37 °C with medium or medium containing NH4Cl (untreated), then treated with RANTES for 10 min at 37 °C (RANTES). After ligand removal, some samples were further incubated in medium containing TAK-779 (+ TAK-779). Cells were stained after permeabilisation for total CCR5 receptors (CCR5) using MC-5, and Ser349 phosphorylated receptors (P-CCR5) using M5/4. Samples were analysed and recorded as described in Figure 1. Bar = 20 μm. C) The experiment illustrated inB was quantitated by counting the proportion of M5/4 positive cells at each time point in medium alone (□) or with NH4Cl (▪). The results are expressed as a percentage of total CCR5 positive cells. A total of 300 cells were counted per sample and the graph shows the mean ± s.d.

To test whether endosomal acidification is required for CCR5 dephosphorylation, we performed the same immunofluorescence experiment in the presence of monensin (not shown) or NH4Cl (Figure 5B, right panels). As for cells treated without the drug, RANTES stimulation led to CCR5 phosphorylation and internalisation. Staining for serine 349 phosphorylated CCR5 was lost with similar kinetics to those seen in the absence of NH4Cl or monensin. Semi-quantitative analysis of M5/4 positive cells indicated that endosomal alkalinisation had no effect on CCR5 dephosphorylation (Figure 5C).

Recycling of β2-AR is pH-dependent

Previous studies with β2-AR have suggested that acidification of early endosomes is important for agonist dissociation, dephosphorylation and resensitisation (20,22). Since we saw only subtle effects of endosomal alkalinisation on CCR5 cycling, we analysed the endocytosis and recycling of β2-AR expressed in CHO cells. CHO HA-β2-AR cells were incubated at 37 °C in suspension with the agonist isoproterenol (20 μm) for up to 30 min. Treatment with agonist was limited to 30 min to prevent the β2-AR degradation seen with longer periods of treatment (7). Internalisation was stopped by cooling and washing the cells on ice, before adding an excess of the antagonist alprenolol (50 μm) and incubating the cells at 37 °C for up to 60 min. When cells were treated with bafilomycin A, monensin or NH4Cl, the drug was added to the cell suspension after the 4 °C washes. HA-β2-AR was then detected by cell surface staining with a rat anti-HA antibody. Figure 6A shows a representative experiment. The internalisation of HA-β2-AR induced by isoproterenol was rapid, but less efficient than that seen for CCR5, with the equivalent of 60% of the initial cell surface pool of receptor remaining at the plasma membrane after 30 min. As soon as the agonist was removed and antagonist added, HA-β2-AR re-appeared at the cell surface, with > 90% of the initial signal recovered by 60 min. However, the kinetics of re-accumulation were slower than those seen with CCR5 (compare Figures 3B, 4 and 6A). Interestingly, for cells incubated with alprenolol in the presence of bafilomycin A, the cell surface fluorescence did not recover with time (Figure 6A). This suggests that in the presence of bafilomycin A, the recycling of HA-β2-AR is inhibited. This experiment was repeated four times using bafilomycin A, monensin or NH4Cl. Despite some variations in the level of receptor down-modulation in the different experiments, we found that all three reagents inhibited the recovery of cell surface receptors during the recycling step (Figure 6B). These results were confirmed by immunofluorescence analysis (data not shown).

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Figure 6. HA-β2-AR recycling is influenced by the pH of endosomes.A) CHO-K1 HA-β2-AR cells were treated in suspension for up to 30 min at 37 °C with isoproterenol (ISO). Following several washes in 4 °C BM (arrow) the cells were incubated at 37 °C for up to 60 min in the presence of alprenolol (ALP) in medium alone (□) or medium containing bafilomycin A (▪). Cell surface HA-β2-AR receptors were detected by indirect immuno-fluorescence with rat anti-HA 3F10 and a biotinylated sheep anti-rat secondary antibody and Streptavidin-FITC. Samples were analysed by flow cytometry. The results are expressed as the percentage of the fluorescence on untreated cells and the graph shows the mean of duplicate values from a representative experiment. B) In addition to medium (□) or bafilomycin A (▪), similar experiments were performed in the presence of monensin(bsl00073), or NH4Cl (bsl00023) during the recycling step. The graph shows the cell surface fluorescence recovery/loss as a percentage of the fluorescence signal on untreated cells. The mean ± s.d. are derived from four separate experiments.

Together, these experiments indicate that as previously proposed, and in contrast to our results with CCR5, the recycling of HA-β2-AR following agonist-induced endocytosis is dependent on transit of the receptors through acidic endosomal compartments and that this recycling can be inhibited by agents raising the pH of endosomes. Moreover, these results show that under conditions in which we failed to see significant effects on CCR5 recycling, the three reagents inhibited β2-AR recycling.

Elution of CCR5-bound chemokines

Our previous results with AOP-RANTES have suggested that, for CCR5, agonist removal is not required for recycling, as receptors on which the ligand-binding site is occupied appear to recycle efficiently (8). To analyse the influence of pH on CCR5–ligand association, we monitored the elution of cell surface-bound radiolabelled RANTES and MIP-1β following incubation of cells in media of increasingly acidic pH. The experiment was performed at 4 °C to prevent ligand internalisation. Radiolabelled chemokines diluted in BM (pH 7.4), were bound to CHO-CCR5 cells, and the cells then washed in media buffered to pH values ranging from 7.4 to 2.0. With both radiolabelled RANTES and MIP-1β, little release of receptor-bound ligand was observed at pH values above 4.0. However, ligand was eluted when cells were incubated in medium with pH values < 4.0, with near complete elution at pH 2.0 (Figure 7). Although we cannot exclude a possible influence of G-protein coupling on the chemokine binding properties, these results suggest that neither RANTES nor MIP-1β dissociate from CCR5 under pH conditions normally found in endosomes (pH > 5.5). In similar experiments with iodinated diferric-transferrin, which is known to bind to its receptor in an acid-sensitive manner (11), ligand elution was observed in the pH range 6–7 (data not shown).

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Figure 7: 125. I-RANTES and 125I-MIP-1β binding to CHO-CCR5 cells. CHO-CCR5 cells were incubated for 90 or 120 min on ice with 125I-RANTES or 125I-MIP-1β in BM pH 7.4, respectively. Labelled cells were then washed with media adjusted to the indicated pH values and the cell-associated radioactivity determined by γ counting. The means and s.d. of triplicate samples for a representative experiment are shown.

pH-dependent degradation of internalised RANTES

Although the release of internalised RANTES and recycling of CCR5 appeared to be independent of exposure to acidic pH, it is nevertheless likely that CCR5 agonists are removed from the receptor during transit through endocytic organelles. The location and mechanism of this dissociation is currently unclear. We investigated the fate of cell-associated RANTES using TCA precipitation assays to detect degradation of the iodinated ligand. 125I-RANTES was bound to CHO-CCR5 cells at 4 °C. Free ligand was washed away and internalisation initiated by rapidly warming the cells to 37 °C. At each time point the cell supernatant was collected and subjected to TCA precipitation to measure intact and degraded RANTES. The cells were also analysed for total cell-associated and acid-resistant (internal) radioactivity. In parallel, we assessed the effect of endosomal alkalinisation on these events by incubating cells in medium containing bafilomycin A, monensin or NH4Cl.

Figure 8 shows a representative experiment for 125I-RANTES internalisation and degradation in the presence or absence of NH4Cl. As suggested by the recycling experiments above, NH4Cl affected neither the rate nor the extent of 125I-RANTES internalisation (Figure 8A). Following warming of the cells to 37 °C, 125I activity was detected in the medium. Much of the material released in the first 10 min after warm-up was TCA-insoluble and presumably represented intact 125I-RANTES dissociated from the cell surface (Figure 8B). Subsequently, TCA-soluble material derived from RANTES degradation accumulated in the medium (Figure 8B). Following internalisation, a decrease in the cell-associated counts was seen due to the release of TCA soluble counts to the supernatant (Figure 8B). Significantly, the release of the TCA soluble activity was inhibited in cells treated with NH4Cl (Figure 8B). Similar results were obtained when cells were treated in the presence of bafilomycin A or monensin, or incubated with 125I-MIP-1β instead of 125I-RANTES (data not shown). This suggests that, although exposure to low pH is not required for RANTES or MIP-1β dissociation, some ligand is removed from the receptor and the degradation of this ligand involves either pH-sensitive proteases or pH-dependent transport to a degradative compartment.

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Figure 8. Internalisation and degradation of 125I-RANTES.125I-RANTES was bound to CHO-CCR5 cells at 4 °C as described in Figure 6. Cells were then warmed to 37 °C for the indicated times in BM alone (open symbols) or BM containing NH4Cl (filled symbols). A) The amount of 125I-RANTES internalised was determined by acid washing at pH 2.0. Each time point indicates the acid-resistant radioactivity (internal) as a proportion of the total cell-associated activity, after subtraction of the background activity at time 0 (▵;▴). B) At the indicated times, the supernatants were collected and subjected to TCA precipitation. The graphs show the amount of cell-associated activity (□;▪), the total radioactivity released in the supernatant (◊;◆), as well as the amount of degraded material (TCA soluble) accumulating in this supernatant with time (○;•). The means and s.d. of triplicate samples from representative experiments are shown.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Reagents

Tissue culture reagents and Nunc tissue culture plastics were from Life Technologies (Invitrogen Ltd, Paisley, UK) and chemicals were from Sigma-Aldrich Company Ltd (Poole, UK) unless otherwise indicated. RANTES and MIP-1β were purchased from R & D Systems Europe Ltd. (Abingdon, UK). 125I-RANTES and 125I-MIP-1β (specific activity 2000 Ci/mmol) were from Amersham Biosciences (Little Chalfont, UK). TAK-779 (18) was obtained through the NIH AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH).

Antibodies

MC-5, a murine monoclonal antibody against human CCR5 was provided by Dr M. Mack (Medizinische Poliklinik, University of Munich, Germany). Purified MC-5 was coupled to Alexa Fluor 488 (488MC-5) using a protein labelling kit (Molecular Probes Europe BV, Leiden, the Netherlands). Monoclonal antibodies against the influenza virus haemagglutinin (HA) (mouse HA.11 and rat 3F10) were purchased from Covance Research Product Inc. (Richmond, CA, USA) and Roche Molecular Biochemicals (Mannheim, Germany), respectively.

Cells

DHFR-deficient Chinese Hamster Ovary (CHO) cells stably expressing human CCR5 (CHO-CCR5) were maintained in nucleoside-free αMEM supplemented with Glutamax and 10% foetal calf serum (FCS), as previously described (9). CHO-K1 cells were maintained in DMEM-F12 containing 10% FCS, 2 mm glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. CHO-K1 cells expressing HA tagged-β2-AR were grown in the same medium supplemented with 1 mg/mL G418. CEMss-CCR5 cells were kept in RPMI supplemented with 10% FCS and 0.2 μg/mL puromycin, as previously described (31).

A pcDNA1 mammalian cell expression construct for the HA-tagged β2-AR (HA-β2-AR) was a gift from Julie Pitcher (MRC-LMCB, UCL, London, UK). CHO-K1 cells were electrophorated with the HA-β2-AR construct together with pSV2-Neo at a ratio of 10 : 1. Transfected cells were selected in medium containing 1 mg/mL G418 and stable HA-β2-AR expressing cell lines isolated by limited dilution.

125I-RANTES and 125I-MIP-1β binding and elution

CHO-CCR5 cells were plated in 16-mm diameter 24-well plates and grown to confluence over 2 days. 125I-RANTES or 125I-MIP-1β (125 pm), diluted in binding medium (BM: RPMI 1640 without bicarbonate containing 0.2% BSA and 10 mm Hepes adjusted to pH 7.0), were added to each well and bound for 90 min (RANTES) or 120 min (MIP-1β) at 4 °C as described (8). Subsequently, unbound ligand was removed by washing twice with ice cold BM. The cells were then rinsed and washed 2 × 5 min with the indicated elution medium. For values between pH 7.4 and 6.0, BM was adjusted to the indicated pH with HCl. For pH 5.0 and below, Hepes was replaced by 10 mm Mes. Following a final wash in cold phosphate-buffered saline (PBS), cells were harvested in 400 μL 0.2 m NaOH and transferred to tubes for γ-counting.

125I-RANTES internalisation and degradation

125I-RANTES (125 pm in BM) was prebound to 85% confluent CHO-CCR5 cells in 16 mm diameter wells at 4 °C. After 90 min, excess ligand was removed by washing with ice cold BM. To allow ligand internalisation, the cold medium was replaced with 1 mL/well 37 °C BM and the plates transferred to a 37 °C incubator. At the indicated time the plates were placed on ice, 700 μL of the medium from each well (SN) collected for TCA precipitation (see below), and the cells washed with cold BM. For each time point, half of the wells were washed with pH 2.0 medium to remove ligand remaining at the cell surface. The cells were harvested in 0.2 m NaOH and the total and acid-resistant cell-associated radioactivity determined by γ-counting. The total amount of radioactivity released in the extracellular medium during the 37 °C incubation was determined by γ-counting 500 μL of the SN fraction. To evaluate the extent of ligand degradation, the remaining 200 μL of each SN sample was made 20% with ice-cold trichloroacetic acid (TCA) and placed on ice. After 1 h the samples were centrifuged at 14 000 r.p.m. for 20 min, and the radioactivity in the supernatant fraction determined by γ-counting.

Analysis of CCR5 distribution by immunofluorescence microscopy

CHO-CCR5 cells grown on coverslips were treated with 125 nm RANTES for 30 or 90 min in 37 °C BM or BM containing bafilomycin, monensin or NH4Cl as indicated. The coverslips were then placed on ice and washed extensively with cold BM. To study receptor recycling, some samples were incubated for a further hour at 37 °C in BM containing 400 nm TAK-779 and the indicated drug. The cells were washed, fixed in 3% paraformaldehyde for 10 min, and free aldehyde groups quenched with 50 mm NH4Cl in PBS. CHO-CCR5 cells were permeabilised in PBS with 0.2% gelatin containing 0.05% saponin, and labelled with 488MC-5 (1.75 μg/mL). After 1 h the cells were washed in PBS with 0.05% saponin, then in PBS and mounted in medium containing Mowiol as described (32). Coverslips were examined using a Zeiss Axioskop microscope, or a Nikon Optiphot-2 microscope equipped with an MRC Bio-Rad 1024 confocal laser scanner. The digital images were assembled using Adobe Photoshop software.

CCR5 endocytosis and recycling

CHO-CCR5 cells (from an 85% confluent 90 mm plate, detached with PBS-EDTA), or CEMss-CCR5 cells (2.5 × 106 per sample), were washed in BM and resuspended on ice in 1 mL of BM or BM containing bafilomycin A, monensin or NH4Cl as indicated. For each condition, RANTES or MIP-1β were added to a final concentration of 125 nm and the cell suspensions transferred to a 37 °C water bath for up to 60 min to allow CCR5 internalisation. Free chemokine was then removed by cooling the samples on ice and washing twice with ice cold BM. The cells were resuspended in BM containing 400 nm TAK-779, together with the indicated drug, and incubated for up to 60 min at 37 °C to allow CCR5 recycling. At the indicated times an aliquot of the cell suspension was taken, transferred to a 96-well plate containing BM and kept on ice. At the end of the experiment the cells in the 96-well plate were washed twice in cold BM. CHO-CCR5 cells were labelled for 90 min on ice with 1.75 μg/mL 488MC-5 in wash buffer, whereas CEMss-CCR5 cells were stained 90 min with MC-5 and then with FITC-conjugated goat antimouse (1/200; Perbio Science UK Ltd, Tattenhall, UK) and rinsed four times in wash buffer (PBS containing 1% FCS and 0.05% azide). The cells were finally resuspended in wash buffer and analysed directly, or after overnight fixation in PBS containing 1% FCS and 1% formaldehyde, using a FACSCalibur flow cytometer (Becton Dickinson UK Ltd, Oxford, UK). For each sample, the Mean Fluorescence Intensity (MFI) was determined from 10 000 accumulated events.

β2- AR endocytosis and recycling

For β2-AR internalisation, CHO-K1 HA-β2-AR cells were treated with 20 μm isoproterenol in BM for up to 30 min. The cells were then washed, as described above, and incubated in BM or BM containing bafilomycin A, monensin or NH4Cl and the antagonist alprenolol (50 μm) for up to 1 h. Cell surface HA-β2-AR receptors were labelled for 90 min on ice with the 3F10 antibody (1 μg/mL) in wash buffer. Cell bound antibody was detected by staining with a biotinylated sheep anti-rat secondary antibody (1 : 100; Amersham Pharmacia Biotech) and Streptavidin-FITC [1/200; Beckman Coulter (UK) Ltd; High Wycombe, UK]. Cells were then washed four times and resuspended in wash buffer before FACS analysis as described above.

SFV infection assay

The recombinant SFV-GFP stock, at 2 × 108 IU/mL, was prepared as previously described (16) and obtained from Derek E. Knight (Kings College London, London, UK). For infection, CHO-CCR5 cells were plated at 6 × 104 cells/well onto coverslips placed in 16 mm wells and cultured for 40 h. Wells were washed in BM and preincubated in 0.3 mL of BM or BM containing monensin, bafilomycin or NH4Cl for 5 min at 37 °C. SFV-GFP was then added to each well to the indicated final concentration and incubated for 90 min at 37 °C. Subsequently, the cell supernatant was aspirated, replaced by fresh tissue culture medium containing 10 μm monensin and the incubation continued for 4.5 h at 37 °C in a CO2 incubator. The cells were then washed and fixed in 3% paraformaldehyde for 10 min. Coverslips were mounted on slides and observed using a Zeiss Axioskop microscope equipped with a Hamamatsu Orca Camera (C4742-95).

Phosphate labelling and CCR5 immunoprecipitation

CHO-CCR5 cells in 3-cm dishes were washed twice with phosphate-free medium (phosphate-free DMEM, 1% BSA and 10 mm Hepes pH 7.5) and incubated with 0.2 mCi of [32P]-orthophosphoric acid for 3 h. The cells were then incubated for up to 10 min in 37 °C phosphate-free medium with or without 80 nm RANTES and then washed three times in medium alone. To analyse CCR5 dephosphorylation, some samples were then further incubated in medium alone or medium supplemented with 200 nm TAK-779. At the end of the incubation, cells were washed twice in ice cold PBS, and lysed in 500 μL of RIPA buffer (1% NP40, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 10 mm NaPi pH 7 and 2 mm EDTA) containing protease inhibitors (1 mm PMSF and 10 μg/mL of chymostatin, leupeptin and pepstatin), and phosphatase inhibitors (10 mm sodium pyrophosphate, 50 mm sodium fluoride, and 1 mm orthovanadate). Detergent-insoluble material was removed by centrifugation at 14 000 r.p.m. for 10 min and the lysates were ‘precleared’ by incubation for 20 min at 4 °C with 30 μL of protein A-Sepharose CL-4B (Amersham Biosciences). To immunoprecipitate CCR5, lysates were mixed for 2 h with protein A-Sepharose CL-4B bound to MC-5 antibody. The beads were collected by centrifugation and washed four times in RIPA buffer, and once in 150 mm NaCl/20 mm Tris buffer. The precipitates were eluted with nonreducing sample buffer and separated on 12% SDS-polyacrylamide gels. Gels were dried and exposed to Kodak (Rochester, NY, USA) X-OMAT AR film for autoradiography.

Detection of CCR5 phosphorylation using a phospho-site specific antibody

M5/4 is a mouse IgG1 monoclonal antibody generated against a peptide encoding the C-terminal 22 amino acids of human CCR5 with phosphoserine residues at the position corresponding to amino acids 336, 337, 342 and 349 as described (21). For immunofluorescence, CHO-CCR5 cells grown on coverslips were pretreated in tissue culture medium alone or containing NH4Cl in 5% CO2 incubator for 30 min at 37 °C. Cells were then stimulated by addition of RANTES (to a final concentration of 125 nm) to the cultures, and incubated for 10 min at 37 °C. For experiments requiring dephosphorylation, samples were subsequently incubated for up to 90 min at 37 °C in medium containing 400 nm TAK-779 with or without NH4Cl or monensin. At the end of the incubations, cells were fixed, quenched and permeabilised as described above. The cells were stained for CCR5 with 488MC-5 and for phosphorylated-CCR5 with M5/4 (hybridoma culture supernatant 1/10) plus a secondary goat antimouse IgG1 coupled to 534Alexa, respectively. Coverslips were washed, mounted and analysed as described above.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

We are grateful to colleagues who have contributed reagents, ideas and discussions to this work. In particular, Matthias Mack for providing the CHO-CCR5 cells and the MC-5 antibody, Derek E. Knight for supplying recombinant SFV-GFP, and Julie Pitcher for helpful discussions and assistance with the β2-AR. We thank Annegret Pelchen-Matthews and Alberto Fraile-Ramos for critical comments on the manuscript. The authors are supported by the UK Medical Research Council.

Note:

During the review of this paper, another study reporting that dissociation of CCR5-bound agonist is pH-independent was published by Weber et al. (33).

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  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
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