Pharmacological characterization of the chemokine receptor, CCR5

Abstract

1. We investigated the effects of a number of naturally occurring chemokines (MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, MCP-4) on different processes linked to the chemokine receptor CCR5 in recombinant CHO cells expressing the receptor at different levels. 2. Internalization of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce internalization (similar50%) of the receptor. Internalization due to MCP-2, MCP-3 and MCP-4 was less (similar20%). 3. Phosphorylation of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce phosphorylation of CCR5 whereas the other chemokines did not induce CCR5 phosphorylation. 4. MIP-1alpha, MIP-1beta, RANTES and MCP-2 were able to stimulate [(35)S]-GTPgammaS binding, an index of receptor/G protein activation, whereas MCP-3 and MCP-4 had no effect in this assay. MCP-2 was a partial agonist (similar80%) compared to MIP-1alpha, MIP-1beta and RANTES, which gave similar maximal stimulations in this assay. 5. MIP-1alpha, MIP-1beta, RANTES, MCP-2 and MCP-4 were able to stimulate increases in intracellular calcium ions via activation of CCR5 whereas MCP-3 was without effect. 6. It is concluded that different chemokines interacting with CCR5 mediate different patterns of cellular responses.

Figure 1

Expression of CCR5 and CD4 on CHO.CCR5 and CHO.CCR5.CD4 cells. Expression was determined using FACS and Western blot analysis as described in the Methods section. (a) FACS analysis for expression of CCR5: black: negative control, bold line: CHO.CCR5 cells, dotted line: CHO.CCR5.CD4 cells. (b) FACS analysis for expression of CD4: black and dotted line: CHO and CHO.CCR5 cells, bold line: CHO.CCR5.CD4 cells. (c) Membranes prepared from the cells were subjected to SDS – PAGE and Western blot analysis using a CCR5 specific antibody (HEK/1/85a/7a): 1: CHO.CCR5 cells, 2: CHO.CCR5.CD4 cells, the same amount of membrane preparation (10 μg) was analysed for both cell types. Arrow indicates CCR5.

Figure 2

Interaction of CD4 and CCR5. Cells were subjected to an immunoprecipitation with an anti-CD4 antibody, separated on SDS – PAGE and transferred to nitrocellulose membrane. CCR5 was detected by ECL using a CCR5 specific antibody (HEK/1/85a/7a) and a corresponding secondary antibody. Experiments on CD4 and CCR5 interaction (two left lanes) were done in CHO.CCR5.CD4 cells and control experiments (right lane) in CHO.CCR5 cells. Arrow indicates CCR5. This is one representative result from three independent experiments performed.

Figure 3

Internalization of CCR5. (a) Time course for CCR5 internalization. CHO.CCR5 cells were harvested in PBS/EDTA and incubated in serum-free medium for 2 h. Cells were then incubated with 50 nM chemokines for various times. After the incubation, the cells were washed in ice-cold PBS +1% FCS +1% NaN₃, and stained for FACS analysis with anti CCR5 HEK/1/85a/7a antibody and anti-rat PE antibody. Internalization rates were determined as described in the Methods section. The graph shows one representative experiment which has been replicated three times. (b) Representative FACS data for an internalization experiment with RANTES, black: negative control, bold line: cells treated with RANTES, dashed line: untreated cells. (c) Dose response experiments for CCR5 internalization. CHO.CCR5.CD4 cells were treated with different amounts of chemokines for 1 h and internalization of the receptor was determined.

Figure 4

Internalization of CCR5 in cells expressing different levels of CCR5. (a) Bars 1 – 4 show data from control CHO.CCR5.CD4 cells and cells that were incubated with 5 mM sodium butyrate for 18 h to increase the receptor density on the cell surface before an internalisation assay was performed as described in the Methods section. Bars 5 and 6 show CHO.CCR5 cells that were treated with MIP-1β alone or MIP-1β incubated with 1 μg of soluble CD4 (sCD4). Data represent the means±range from two independent experiments performed (**P<0.05 relative to control). (b) Bars 1 and 3 show HeLa RC-4 cells treated with MIP-1α and MIP-1β respectively, bars 2 and 4 show treatment of HeLa RC-49 cells. (c) (1) FACS analysis to determine the cell surface expression of CCR5 in CHO.CCR5.CD4 cells. black: negative control, thin line: CHO.CCR5.CD4 cells (mean fluorescence: 47.14), bold line: CHO.CCR5.CD4 cells treated with sodium butyrate (mean fluorescence: 89). (2) FACS analysis to determine the cell surface expression of CD4, black CHO.CCR5.CD4 cells (mean fluorescence: 21.77), bold line: CHO.CCR5.CD4 cells treated with sodium butyrate as described (mean fluorescence: 69.53). (3) FACS analysis to determine the cell surface expression of CCR5 in HeLa RC-4 cells: black: negative control, thin line HeLa RC-4 (mean fluorescence 45.7). (4) FACS analysis to determine the cell surface expression of CCR5 in HeLa RC-49 cells: black: negative control, thin line HeLa RC-49 (mean fluorescence 171.6).

Figure 5

Effects of chemokines on CCR5 determined using confocal microscopy. Cells were grown on coverslips overnight, incubated in medium without serum for 2 h and then incubated with 50 nM chemokines or vehicle control for 1 h (a) control, (b) MIP-1α, (c) MIP-1β, (d) RANTES. Cells were washed and a stain was performed using anti-CCR5 HEK/1/85a/7a antibody and anti-rat-FITC. Pictures were taken using Leica NT Confocal Imaging system. Data show representative cells.

Figure 6

Detection of chemokine induced phosphorylation in CHO.CCR5 cells. Cells were incubated in medium without serum and incubated with chemokines (50 nM) as indicated and then subjected to an immunoprecipitation with an anti CCR5 antibody. CCR5 was detected on a Western blot using a specific antibody. Arrows indicate CCR5. (a) MIP-1α treatment at different time points. Results for MIP-1β and RANTES treatment were similar (data not shown). (b,c) Treatment with protein phosphatase 1. Results for MIP-1α were similar (data not shown). Data show one representative blot from three independent experiments with similar results.

Figure 7

Stimulation of [³⁵S]-GTPγS binding in CHO.CCR5 and CHO.CCR5.CD4 membranes by natural chemokines. [³⁵S]-GTPγS binding was determined after 30 min incubation in the presence of increasing concentrations of each agent as described in the Methods section. Results are expressed as a percentage of the unstimulated basal level of binding. Data shown are the means of triplicate determination from a representative of at least three separate experiments. Summary data are provided in Table 1. (a) Stimulation in CHO.CCR5.CD4 cells induced by MIP-1α, MIP-1β and RANTES. (b) Stimulation in CHO.CCR5 cells induced by RANTES, MCP-2, MCP-3, MCP-4 (closed symbols) and CHO.CCR5 cells after PTX (15 h) treatment (open symbols).

Figure 8

Intracellular calcium ion concentrations in CHO.CCR5.CD4 cells. Intracellular calcium ion concentrations were determined after stimulation of CCR5 with the indicated chemokines as described in the Methods section. Data are expressed as mean±s.e.mean (three or more experiments).