Endocytosis and recycling of the HIV coreceptor CCR5 - PubMed (original) (raw)

Endocytosis and recycling of the HIV coreceptor CCR5

N Signoret et al. J Cell Biol. 2000.

Abstract

The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect. Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.

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Figures

Figure 1

CCR5 downmodulation and recycling. Duplicate sets of CHO-CCR5 cells were treated with RANTES (500 nM, white bars) or AOP-RANTES (100 nM, black bars) for 1 h at 37°C or were left untreated (stippled bar). One set of treated cells was cooled to 4°C, whereas the other was washed and incubated in the absence of ligand for a further 1 h at 37°C. Subsequently, all cells were placed on ice and incubated for 2 h at 4°C with 125I–MC-5. Cell-associated radioactivity was determined as described in Materials and Methods. The cell-associated counts are expressed as a percent of the untreated controls. The means and standard deviations for quadruplicate samples are shown for a representative experiment.

Figure 4

Localization of downmodulated CCR5. CHO-CCR5 cells were treated with RANTES (500 nM) for 4 h at 37°C. After fixation and permeabilization, the cells were stained for CCR5 and the lysosomal markers LBPA (A) and Lgp-B (B), respectively. Open arrowheads indicate CCR5-positive, but LBPA- or Lgp-B–negative, vesicles; white arrowheads indicate LBPA- (A) or Lgp-B–positive (B), but CCR5-negative, late endocytic structures. Bar, 10 μm.

Figure 5

Ultrastructural localization of CCR5. CHO-CCR5 cells were pulsed with BSA–gold (5 nM, BSA–G5) for 20 min and chased with medium for 4 h to mark late endocytic organelles. Cells were treated with 500 nM RANTES (A and D) or 125 nM AOP-RANTES (B, C, E, and F) during the last 2 h of this chase, washed, and processed for cryosection immunolabeling EM. CCR5 was detected by staining with MC-5 and 10 nm (B) or 15 nm (A and C–F) protein A–gold particles. cp, coated pit; G, Golgi complex. C–F show double label staining of sections for CCR5 (15 nm gold particles, large arrows) with the markers of the endocytic pathway Lgp-B (C), CD63 (D), or the TfR (E and F) labeled with 10 nM gold particles (small arrows). BSA–G5–containing late endocytic vacuoles are marked with an asterisk. The CCR5-containing tubules and vesicles are distinct from the organelles labeled with Lgp-B (C) or CD63 (D), even though these markers can occasionally be observed in the same part of the cell. In contrast, the TfR-containing tubules are intermingled with MC-5–labeled tubules and vesicles (E and F), with the large and small gold particles are occasionally found in the same vesicles. Note the prominent coats on some of the CCR5-containing vesicles (open arrowheads in C–E). Bars, 200 nm.

Figure 3

CCR5 turnover in ligand-treated CHO cells. (A) CHO-CCR5 cells were incubated in BM or BM-containing CHX for up to 9 h at 37°C. One tenth of each cell lysate was loaded per lane and Western blot analysis was performed using 125I–MC-5. Antibody binding was determined for each band and is illustrated as the mean ± SD for three independent experiments, and one representative blot is depicted. 9− indicates cells incubated in BM for 9 h without CHX. (B) Cells were incubated in BM plus CHX (white bars) or BM/CHX containing 500 nM RANTES (gray bars) or 100 nM AOP-RANTES (black bars) for 1 or 6 h and the amount of cellular CCR5 was analyzed as described in A.

Figure 3

Figure 6

Recycling of CCR5. (A) CHO-CCR5 cells were incubated in BM or BM with 100 nM RANTES or 100 nM AOP-RANTES for 1 h at 37°C. Cells were then fixed and stained with MC-5 and a FITC-conjugated goat anti–mouse antibody. Cells were stained intact to show cell surface CCR5 or after permeabilization to show intracellular plus cell surface CCR5 (Total). (B) CHO-CCR5 cells were initially treated with RANTES or AOP-RANTES as in A, but after downmodulation, the cells were washed extensively at 4°C in BM, and then incubated in BM containing MC-5 for 1 h at 37°C. The cells were then fixed and stained intact, or after permeabilization with Saponin, with the FITC-conjugated anti–mouse antibody. Bar, 20 μm.

Figure 7

Quantitative analysis of 125I–MC-5 feeding. CHO-CCR5 cells were incubated in BM (white bars, and □) or BM containing RANTES (500 nM, gray bars and •) or AOP-RANTES (100 nM, black bars and ▴) for 1 h at 37°C. CCR5 molecules remaining at the plasma membrane were then labeled on ice with 125I–MC-5 to determine the extent of downmodulation. After this saturation step, some cells were reincubated in prewarmed medium containing 125I–MC-5 to allow iodinated antibody to label recycling CCR5. (A) Cells were fed with the 125I–MC-5 for 1 h in the presence or absence of CHX. The graph indicates the cell-associated radioactivity for each point as a percent of the radioactivity bound on untreated cells and is the mean ± SD for triplicate samples. (B) To determine the kinetics of 125I–MC-5 uptake, cells were fed with 125I–MC-5 for various times after washing out RANTES or AOP-RANTES. The graph shows the relative increase in cell-associated radioactivity at each time point, calculated by dividing the total cell-associated radioactivity at each time point by the cell-associated activity at time zero. Each point represents the mean ± SD of triplicate samples from a representative experiment.

Figure 2

Binding and internalization of iodinated RANTES and AOP-RANTES. (A) CHO-CCR5 cells were incubated with 125 pM 125I-RANTES or 125I–AOP-RANTES for 90 min at 4°C. Bound-radiolabeled ligands (black bars) were eluted from the cell surface by washes at pH 2 (white bars) or pH 11.6 (stippled bars). The means and standard deviations for triplicate samples are shown for a representative experiment. (B) 125I-RANTES (closed symbols) and 125I–AOP-RANTES (open symbols) were bound to CHO-CCR5 (▪ and □) or CHO-K1 (•, ○) cells as described in A. Cells were then warmed to 37°C for the indicated times, and the ligand remaining on the cell surface was removed by treatment with pH 11.6 buffer at 4°C, as decribed in A. Each time point indicates the alkaline resistant (internal) radioactivity as a proportion of the total cell-associated activity, after subtraction of the background activity at time zero.

Figure 2

Figure 8

Downmodulation and recycling of CCR5 on CHO cells deficient in GAG synthesis. (A) CHO-pgsA-745–expressing CCR5 (medium) were treated in suspension with 100 nM RANTES or 100 nM AOP-RANTES for 1 h (ligand). After washout of the chemokines, some cells were incubated in fresh medium for a further 1 h (ligand, washout). Cells were then fixed and stained with MC-5 and FITC-conjugated anti–mouse antibody and the level of cell-surface fluorescence was measured by flow cytometry. Cells labeled with the secondary antibody only were used as a negative control (shaded peak). (B) CHO-pgsA-745-CCR5 cells were incubated in medium alone (medium) or medium containing 100 nM RANTES for 1 h (RANTES). Excess chemokine was removed by washing and the cells were incubated in fresh medium for one more hour (RANTES, washout). Cells were then treated for a second time with medium containing 100 nM RANTES for 1 h (RANTES, washout, RANTES). Finally, cells were fixed, stained, and analyzed, as described in A. (C) CHO-pgsA-745-CCR5 cells were incubated in medium alone (left) or medium containing 100 nM AOP-RANTES (right) for 1 h. Excess chemokine was removed by washing and then the cells were fed with MC-5 for 1 h at 37°C. Cells were fixed, permeabilized, and stained with the FITC-conjugated anti–mouse antibody. Bar, 20 μm.

Figure 8

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Endocytosis and recycling of the HIV coreceptor CCR5 - PubMed (original) (raw)