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J Biol Chem, Vol. 275, Issue 13, 9201-9208, March 31, 2000

Regulation of the Human Chemokine Receptor CCR1
CROSS-REGULATION BY CXCR1 AND CXCR2^*

Ricardo M. Richardson^§, Bryan C. Pridgen, Bodduluri Haribabu, and Ralph Snyderman^¶

From the Departments of  Medicine and ^¶ Immunology, Duke University Medical Center, Durham, North Carolina 27710

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca²⁺ mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca²⁺ mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (CCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca²⁺ mobilization. S2, S3, and CCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca²⁺ mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca²⁺ mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca²⁺ mobilization by S3 and CCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and CCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca²⁺ mobilization) which is inversely corrolated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RANTES¹ is a member of the CC subfamily of chemokines, which participate in the recruitment and activation of leukocytes (1-3). RANTES interacts with specific cell surface receptors, which are coupled to pertussis toxin-sensitive guanine nucleotide regulatory proteins (G protein) to activate effectors such as phospholipase C (PLC), ion channels, phospholipase D, and protein kinase C (4-6). In addition to the CCR1 receptor, RANTES activates several members of the CC subfamily of chemokine receptors including CCR3, CCR4, and CCR5 (2, 3). CCR1 is also activated by MIP-1, MCP-2, and MCP-3, although maximum responses are only obtained with RANTES and MIP-1 (2, 3, 7-9).

While much has been learned about the signaling pathways of chemokine receptors, little is known about their mechanism(s) of regulation or cross-regulation. Cellular responses to chemoattractants such as formyl peptides (fMLP), a complement cleavage product (C5a), interleukin-8 (IL-8), platelet-activating factor, monocyte chemoattractant protein-1 (MCP-1) and leukotriene B₄, are regulated via three forms of desensitization: 1) desensitization that involves receptor/G protein uncoupling via phosphorylation of the activated receptor by a receptor-specific kinase (GRK) (10-15), 2) desensitization that occurs via phosphorylation of receptors by a second messenger activated kinase (10-15), and 3) downstream inhibition of PLC activation among groups of chemoattractant receptors (16). This present work studied the mechanisms of regulation and cross-regulation of CCR1. For that purpose, an epitope-tagged CCR1 was expressed in a rat basophilic leukemia (RBL-2H3) cell line and studied for its ability to undergo phosphorylation and desensitization upon agonist stimulation. In addition, CCR1 was co-expressed with the receptors for IL-8 (CXCR1 and CXCR2), and its ability to undergo or mediate cross-desensitization was investigated. The results show that CCR1 is regulated via receptor phosphorylation as well as a phosphorylation-independent mechanism. In addition, the data demonstrated unexpected differences in the ability of CCR1 to cross-regulate cellular responses to CXCR1 and CXCR2. These differences likely reflect the disparate susceptibility of CXCR1 and CXCR2 to time-dependent receptor signals from CCR1. More broadly, these data suggest that cross-regulation among classes of chemoattractant receptors is dependent on the strength of receptor's signaling, which may be inversely correlated with the receptor's susceptibility to phosphorylation by second messenger dependent kinases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [³²P]Orthophosphate (8500-9120 Ci/mmol), myo-[2-³H]inositol (24.4 Ci/mmol), and [-³²P]GTP (6000 Ci/mmol) were purchased from NEN Life Science Products. ¹²⁵I-RANTES and ¹²⁵I-IL-8 were obtained from Amersham Pharmacia Biotech. RANTES, IL-8 (monocyte-derived), MIP-1, and MCP-2 were purchased from Genzyme. Geneticin (G418) and all tissue culture reagents were purchased from Life Technologies, Inc. Monoclonal 12CA5 antibody, protein G-agarose and protease inhibitors were purchased from Roche Molecular Biochemicals. Anti-human IL-8RB (CXCR2) antibody was purchased from PharMingen. Polyclonal antibody against PLC₃ and anti-human IIL-8RA (CXCR1) antibody were obtained from Santa Cruz Biotechnology. Indo-1 acetoxymethyl ester and pluronic acid were purchased from Molecular Probes. 8-(4-Chlorophenylthio)-adenosine 3'-cyclic monophosphate (cpt-cAMP), phorbol 12-myristate 13-acetate (PMA), GDP, GTP, and ATP were purchased from Sigma. All other reagents are from commercial sources. The cDNAs encoding the hemagglutinin (HA) epitope-tagged CCR1 and the CXCR2 mutant 331T were kindly provided by Dr. Timothy N. C. Wells and Dr. Ann Richmond, respectively.

Construction of the Phosphorylation-deficient Mutants of CCR1-- The polymerase chain reaction was used to generate phosphorylation-deficient mutants of CCR1 (S1, S2, and S3) as well as a carboxyl-terminal truncated CCR1 mutant (CCR1). The 5' oligonucleotide corresponding to the epitope-tagged CCR1 (YPYDVPDYA) was used with a 3' oligonucleotide complementary to the CCR1 tail replacing serine and threonine residues with alanine (S1, S2, and S3) or to the amino acids 325-331 in CCR1 following by a stop codon (CCR1). The resulting polymerase chain reaction products were cloned into the eukaryotic expression vector pcDNA3, and the entire receptors were sequenced to confirm the intended mutations and lack of secondary mutations.

Cell Culture and Transfection-- RBL-2H3 cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml) (17). RBL-2H3 cells (1 × 10⁷ cells) were transfected by electroporation with pcDNA3 containing the receptor cDNAs (20 µg), and Geneticin-resistant cells were cloned into single cell by flow cytometry (fluorescence-activated cell sorting) analysis. For double transfectant, RBL cells stably expressing wild type CCR1 or the carboxyl-terminal deletion mutant CCR1 were electroporated with pRK5 plasmid containing either CXCR1 or CXCR2. The following day cells were analyzed by fluorescence-activated cell sorting for cell surface expression of the receptors, using specific antibodies against the amino terminus of either CXCR1 or CXCR2. The top 3% of the cells expressing the receptors were subjected to two runs of sorting and then cloned into single cell. Cells expressing similar number of both receptors were used in this study.

Radioligand Binding Assays-- RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 × 10⁶ cells/well) in growth medium. Cells were then rinsed with Dulbecco's modified Eagle's medium supplemented with 20 mM Hepes, pH 7.4, and 10 mg/ml bovine serum albumin and incubated on ice for 2 h in the same medium (200 µl) containing the radiolabeled ligand (0.1 nM). Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline containing 10 mg/ml bovine serum albumin, and washed three times with the same buffer. Cells were then lysed with 200 µl of radioimmune precipitation buffer, and bound radioactivity was counted (18, 19). Nonspecific radioactivity bound was determined in the presence of 1 µM unlabeled ligand. For internalization, cells were incubated with different ligands for 0-60 min. Then cells were washed with phosphate-buffered saline, and ¹²⁵I-RANTES or ¹²⁵I-IL-8 binding was carried out as described above.

Phosphoinositide Hydrolysis and Calcium Measurement-- RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed fetal bovine serum and 1 µCi/ml [³H]inositol. The generation of inositol phosphates was determined as reported (17). For calcium mobilization, cells (3 × 10⁶) were removed, washed with HEPES-buffered saline, and loaded with 1 µM Indo I-AM in the presence of 1 µM pluronic acid for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. Intracellular calcium increase in the presence and absence of ligands was measured as described (17).

Phosphorylation of Receptors and PLC₃-- Phosphorylation of receptors or PLC₃ was performed as described previously (17-19). RBL (5 × 10⁶) expressing the receptors were incubated with [³²P]orthophosphate (150 µCi/dish) for 90 min. Then labeled cells were stimulated with the indicated ligands for 5 min at 37 °C. Cell lysates were immunoprecipitated with specific antibodies against either the NH₂ terminus of CXCR1 or CXCR2; the HA epitope tag of CCR1 or the PLC₃, analyzed by SDS electrophoresis and visualized by autoradiography.

GTPase Activity-- Cells were treated with appropriate concentrations of stimulants, and membranes were prepared as described previously (17). GTPase activity using 10-20 µg of membrane preparations was carried out as described previously (18, 19).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Characterization of CCR1 in RBL-2H3 Cells-- Competition binding assays using ¹²⁵I-RANTES (Fig. 1A) and Scatchard analysis (data not shown) revealed that the CCR1 stably expressed in RBL-2H3 cells bound RANTES with a dissociation constant (K_d) of 12 ± 2 nM and B_max of 7354 ± 462 receptors/cell. The K_d for RANTES binding in RBL-2H3 cells is similar to that of the CCR1 expressed in 293 cells 7.6 ± 1.5 nM (4). Activation of CCR1 by RANTES stimulated PI hydrolysis (Fig. 1B) and secretion (Fig. 1D) in a dose-dependent manner in RBL-2H3 cells. The EC₅₀ values for RANTES were 2.17 ± 0.25 nM and 3.3 ± 0.65 nM for PI hydrolysis and exocytosis, respectively. CCR1 mediated comparable peak intracellular Ca²⁺ mobilization in response to both RANTES and MIP-1 (Fig. 1C). MCP-2-induced Ca²⁺ mobilization was ~50% less than RANTES and MIP-1. No response was obtained with IL-8. Pretreatment of the cells with pertussis toxin completely inhibited the ability of all three ligands tested to stimulate PI hydrolysis, Ca²⁺ mobilization, and secretion (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Characterization of CCR1 expressed in RBL-2H3 cells. A, for competition binding RBL cells (5 × 105) expressing CCR1 were incubated with 125I-RANTES in the presence of different concentrations of unlabeled ligand at 4 °C for 2 h. The values are presented as percentage of total, which is defined as the total amount of 125I-RANTES bound to control (no cold ligand added) cells. The experiment was repeated twice with similar results. B, for the generation of inositol phosphates ([3H]IPs), cells were cultured overnight in the presence of [3H]inositol (1 µC/ml). Cells were preincubated (10 min, 37 °C) with a HEPES-buffered saline containing 10 mM LiCl in a total volume of 50 µl and stimulated with different concentrations of RANTES for 10 min. Supernatant was used to determine the release of [3H]IPs. Data was corrected for basal and represented as total counts/min. The experiment was repeated four times with similar results. C, for intracellular calcium mobilization, RBL cells (5 × 106) were loaded with Indo-1 and RANTES, MCP-2, MIP-1, and IL-8 (10 nM) stimulated Ca2+ mobilization were measured. Representative tracings of five experiments are shown. D, for secretion, 15 µl of the supernatant for PI hydrolysis was removed and -hexosaminidase release was measured. Data are represented as percentage of total -hexosaminidase in the cells. The experiment was repeated four times with similar results.

Desensitization of CCR1 in RBL-2H3 Cells-- Ca²⁺ mobilization was quantified to study desensitization of CCR1-mediated cellular responses in RBL. As shown in Fig. 2, prestimulation of Indo-1-loaded RBL cells expressing CCR1 with an EC₁₀₀ dose (for Ca²⁺ mobilization) of RANTES (10 nM), MIP-1 (10 nM), or MCP-2 (10 nM) markedly inhibited response to a second dose of RANTES. Desensitization by RANTES and MIP-1 was >90%, whereas MCP-2 pretreatment caused ~50% desensitization. PMA (100 nM) and cpt-cAMP (1 mM) pretreatment of the cells completely inhibited RANTES-, MIP-1-, and MCP-2-induced Ca²⁺ mobilization (Fig. 2, data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Desensitization of CCR1-mediated intracellular calcium mobilization in RBL-2H3 cells. RBL-2H3 expressing CCR1 were loaded with the calcium indicator Indo-1 and exposed to a first EC100 dose of RANTES, MIP-1, MCP-2, PMA, or cpt-cAMP. Cells were rechallenged 3 min later with a second dose of ligand as indicated. Traces are representative of three experiments.

Pretreatment of CCR1-expressing RBL cells with either RANTES (100 nM) or PMA (100 nM) produced ~60% desensitization of RANTES-mediated GTPase activity in membranes compared with control or untreated cells (Fig. 3). In contrast to Ca²⁺ mobilization (Fig. 2), pretreatment with cpt-cAMP (1 mM) had no effect on RANTES-stimulated GTPase activity (Fig. 3).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Desensitization of CCR1-mediated GTPase activity. RBL-2H3 cells expressing CCR1 were treated with RANTES (100 nM), PMA (100 nM) or cpt-cAMP (1 mM) for 5 min. Membranes were prepared and assayed for agonist-stimulated GTP hydrolysis. The data are presented as percentage of control, which is the net maximal stimulation obtained with untreated cells. Data shown are representative of one of three experiments performed in triplicate.

Phosphorylation of CCR1-- RANTES (Fig. 4A, lane 2), MIP-1 (lane 5), and MCP-2 (lane 6) induced homologous phosphorylation of CCR1 (~50 kDa). MCP-2-mediated phosphorylation was less than that of RANTES and MIP-1. Heterologous phosphorylation by PMA (lane 3) was also lower (~50% less) than that of RANTES and MIP-1. No phosphorylation of CCR1 was detected with cpt-cAMP (lane 4).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of CCR1. A, 32P-labeled RBL-2H3 cells (5 × 106/60-mm plate) expressing CCR1 were incubated for 5 min with (lanes 2-5) or without (lane 1) stimulants. Cells were lysed, immunoprecipitated with 12CA5 antibody, and analyzed by SDS-PAGE and autoradiography. B, 32P-labeled CCR1 cells were incubated with and without staurosporine for 5 min and then stimulated with either RANTES (lanes 3 and 4), MCP-2 (lanes 5 and 6), or PMA (lanes 7 and 8). Cells were lysed, immunoprecipitated with 12CA5 antibody, electrophoresed into 10% SDS-polyacrylamide gel, and autoradiographed. Three other experiments yielded similar results.

Homologous phosphorylation of CCR1 by RANTES (Fig. 4B, lanes 3 and 4) or MCP-2 (lanes 5 and 6) was partially inhibited by pretreatment of the cells with the protein kinase C inhibitor staurosporine (100 nM). PMA-induced heterologous phosphorylation of CCR1 was totally blocked by staurosporine (lanes 7 and 8). These results suggest that CCR1 is susceptible to phosphorylation by a GRK- and a protein kinase C-dependent mechanism.

IL-8-mediated Cross-desensitization of CCR1-- It was determined whether CCR1-mediated cellular responses are regulated by cross-desensitization. Double transfectant RBL cells expressing CCR1 (7354 ± 462 receptors/cell) and the receptors for IL-8, CXCR1 (7009 ± 131 receptors/cell), CXCR1-CCR1, or CXCR2 (7452 ± 387 receptors/cell), CXCR2-CCR1, were constructed. The K_d for CXCR1 (1.19 ± 0.69 nM) and CXCR2 (3 ± 0.73 nM) in RBL co-expressing CCR1 were similar to that of RBL cells expressing single receptor (19). RANTES- and MIP-1-mediated Ca²⁺ mobilization by CCR1 was cross-desensitized (~65%) by CXCR1 or CXCR2 upon pretreatment of the cells with IL-8 (Table I). Pretreatment of the cells with a first dose of either RANTES or MIP-1 diminished Ca²⁺ mobilization by CXCR2 (~35%). CXCR1 was not desensitized by CCR1. In cells expressing CCR1 and a phosphorylation-deficient mutant of CXCR2, 331T (331T-CCR1), IL-8-mediated Ca²⁺ mobilization was still cross-desensitized by RANTES and MIP1- and vice versa (Table I).

View this table: [in this window] [in a new window]   Table I Cross-desensitization of CCR1-mediated Ca2+ mobilization by the receptors for IL-8 CXCR1, CXCR2, and the CXCR2 mutant 331T RBL-2H3 cells (5 × 106 cells/assay) expressing CXCR1-CCR1, CXCR2-CCR1, or 331T-CCR1 were loaded with Indo-1 and stimulated with RANTES (10 nM), MIP-1 (10 nM), or IL-8 (10 nM). Cells were rechallenged 3 min later with a second dose of the indicated ligand, and peak intracellular Ca2+ mobilization was determined. Data are the means ± S.E. of three different experiments.

Pretreatment of CXCR2-CCR1 cells with RANTES (100 nM) inhibited GTPase activity mediated by both RANTES (~50%) and IL-8 (~40%) in membrane (Fig. 5). IL-8 pretreatment also inhibited GTPase activity in response to both IL-8 (~50%) and RANTES (~40%). Pretreatment with IL-8 for 5 min caused ~50% desensitization of IL-8-mediated GTPase activity in membranes, whereas ~90% of CXCR2 are internalized after 1-2 min of exposure of the cells to IL-8 (Ref. 19, Fig. 7). Two factors may account for this difference: loss of phosphates group by part of the receptors during membrane preparation or recovery of internalized (i.e. non-desensitized) receptors from membranes vesicules.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Cross-desensitization of CCR1-mediated GTPase activity. Double transfected RBL-2H3 cells expressing CCR1 and CXCR2 (CXCR2-CCR1) were treated with IL-8 (100 nM) or RANTES (100 nM) for 5 min. Membranes were prepared and assayed for agonist-stimulated GTP hydrolysis. The data shown are the means of three different experiments performed in triplicate. The data are presented as percentage of control, which is the net maximal stimulation obtained with untreated cells. Data shown are representative of one of three experiments performed in triplicate.

Cross-phosphorylation of CCR1, CXCR1, and CXCR2-- CXCR1-CCR1 and CXCR2-CCR1 RBL cells were ³²P-labeled and stimulated with either RANTES (100 nM) or IL-8 (100 nM). The cell lysates were immunoprecipitated first with the 12CA5 antibody specific for the HA epitope tag expressed at the NH₂ terminus of CCR1 and then with specific antibodies directed against the NH₂ terminus of CXCR1 or CXCR2 (20). CCR1 was homologously phosphorylated by RANTES (Fig. 6, A and B, lanes 5) and cross-phosphorylated upon stimulation by IL-8 of either CXCR1 (Fig. 6A, lane 6) or CXCR2 (Fig. 6B, lane 6). CXCR1 and CXCR2 were homologously phosphorylated by IL-8 (Fig. 6, A and B, lanes 3). CXCR2, but not CXCR1, was cross-phosphorylated by RANTES stimulation of CCR1 (Fig. 2, compare lane 2 in panel B with lane 2 in panel A).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Cross-phosphorylation of CCR1 by the IL-8 receptors CXCR1 and CXCR2. 32P-Labeled RBL-2H3 cells (5 × 106/60-mm plate) expressing CCR1 with either CXCR1 (CXCR1-CCR1) or CXCR2 (CXCR2-CCR1) were incubated for 5 min with or without stimulants as shown. Cells were lysed, immunoprecipitated first with 12CA5 antibody and then CXCR1- or CXCR2-specific antibody, analyzed by SDS-PAGE, and autoradiographed. The results are from a representative experiment that was repeated five times.

Internalization of CCR1-- CCR1 undergo rapid receptor internalization (t_1/2 ~7 min) upon exposure of either CXCR1-CCR1- or CXCR2-CCR1-expressing cells to 100 nM RANTES (Fig. 7, panels A and C). Maximum internalization was ~90% after 60 min. RANTES had no effect on CXCR1 or CXCR2 (Fig. 7, panels B and D). IL-8 caused ~65% and ~95% internalization of CXCR1 and CXCR2, respectively (Fig. 7, panels B and D) but showed no significant decrease in ¹²⁵I-RANTES binding to CCR1 on either CXCR1-CCR1 or CXCR2-CCR1 cells (Fig. 7, panels A and C). Despite inducing CCR1 cross-phosphorylation, treatment of double transfected cells with IL-8 did not cause CCR1 internalization, indicating cross-phosphorylation does not stimulate this process.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Internalization of CCR1 in RBL cells. CXCR1-CCR1 or CXCR2-CCR1 cells (0.5 × 106 cells/well) were incubated with either RANTES (100 nM) or IL-8 (100 nM) for 0-60 min. Cells were then washed and assayed for 125I-RANTES (A and C) or 125I-IL-8 (B and D) binding. The values are presented as percentage of total, which is defined as the total amount of 125I-ligand bound to control (untreated) cells. The experiment was repeated four times with similar results.

Expression and Characterization of CCR1 Mutants in RBL-2H3 Cells-- In order to assess the role of phosphorylation in the desensitization CCR1, four receptor mutants lacking specific serine and threonine residues were constructed (Table II) and stably expressed into RBL-2H3 cells. Competition binding using ¹²⁵I-RANTES and Scatchard analysis indicated that the pharmacological properties of the mutants are similar to that of the wild type CCR1 (Table III). The differences in K_d observed between the mutants and the wild type CCR1 (Table III) are not statistically significant (p > 0.05), as determined by paired test. Clones expressing similar receptor numbers (Table III) were utilized to determine the functional properties of the mutants CCR1 versus the wild type receptor. S2, S3, and CCR1 were more active than CCR1 and S1 in mediating RANTES-induced PI hydrolysis (Fig. 8A). Peaks of intracellular Ca²⁺ mobilization in response to RANTES (10 nM) were similar for wild type and mutants CCR1 (Fig. 8B). However, a more sustained response was obtained with S2, S3, and CCR1 as compared with CCR1 and S1. S2, S3, and CCR1 were resistant to RANTES-induced receptor internalization, relative to CCR1 and S1 (Fig. 8C).

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Functional characteristics of the phosphorylation deficient mutants of CCR1. A, phosphoinositide hydrolysis in RBL-2H3 cells expressing CCR1, S1, S2, S3, and 41 CCR1 were determined as described in the legend to Fig. 1. Data are represented as -fold stimulation over basal. Results shown are representative of one of three experiments performed in triplicate. B, RANTES (10 nM) stimulated intracellular calcium mobilization were measured as described in legend to Fig. 2. Representative tracings of four experiments are shown. C, internalization of CCR1, S1, S2, S3, and CCR1 was measured as described in legend for Fig. 7. The data are representative of two experiments performed in triplicate. D, 32P-labeled RBL-2H3 cells (5 × 106) expressing the CCR1, S1, S2, S3, and CCR1 were stimulated with RANTES or PMA (100 nM). Receptor phosphorylation was determined as described in the legend of Fig. 4. The results are from a representative experiment that was repeated three times.

S2, S3, and CCR1 were also resistant to RANTES and PMA-mediated receptor phosphorylation (Fig. 8D) and desensitization of GTPase activity in membranes (Fig. 9A). The resistance of S2 to phosphorylation relative to S1 and CCR1 indicate that the phosphorylation sites for CCR1 are located in the cluster of serine and threonine which comprises amino acids 340-346. Ca²⁺ mobilization in response to RANTES was desensitized by pretreatment of the cells to a first dose of RANTES (Fig. 9B). However, S2, S3, and CCR1 (~67%, ~59%, and 61%, respectively) were more resistant to desensitization than S1 and CCR1 (~82% and 86%, respectively).

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Desensitization of the phosphorylation-deficient mutants of CCR1. A, CCR1-, S1-, S2-, S3-, and CCR1-expressing cells were treated with RANTES (100 nM) or PMA (100 nM). Membranes were prepared, and GTPase activity was measured as described in the legend of Fig. 3. Data shown are representative of one of three experiments performed in triplicate. B, RBL cells (5 × 106) were loaded with Indo-1 and RANTES (10 nM)-stimulated Ca2+ mobilization was measured. Data are represented as percentage of desensitization of the response obtained with the first dose of RANTES. This experiment was repeated three times with similar results.

Cross-phosphorylation and Cross-desensitization of S3 and CCR1-- RBL-2H3 cells co-expressing the CCR1 mutant S3 (8031 ± 603 receptors/cell) and CXCR1 (6585 ± 911 receptors/cell), (S3-CXCR1) or CXCR2 (6890 ± 511 receptors/cell), (S3-CXCR2); or cells expressing CCR1 (7273 ± 312 receptors/cell) and CXCR1 (6930 ± 107 receptors/cell), CCR1-CXCR1, or CXCR2 (7121 ± 539 receptors/cell), CCR1-CXCR2, were generated to determine the role of receptor cross-phosphorylation on CCR1 cross-desensitization. S3 and CCR1 were resistant to cross-phosphorylation by both CXCR1 and CXCR2 upon IL-8 activation (data not shown). In contrast to CCR1, RANTES stimulation of either S3 or CCR1 resulted in cross-phosphorylation of both CXCR1 (Fig. 10, lanes 2 and 6) and CXCR2 (data not shown). CXCR1 was also homologously phosphorylated by IL-8 (Fig. 10, lanes 3 and 5). S3 and CCR1 activation by RANTES caused no internalization of either CXCR1 or CXCR2 (data not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 10.   Cross-phosphorylation among S3, CCR1, and CXCR1. 32P-Labeled S3-CXCR1 and CCR1-CXCR1 cells (5 × 106/60-mm plate) were stimulated for 5 min with RANTES (100 nM) or IL-8 (100 nM) and immunoprecipitated with anti-CXCR1 antibody. Receptor phosphorylation was determined as described in the legend of Fig. 6. The results are from a representative experiment that was repeated three times.

S3- and CCR1-mediated Ca²⁺ mobilization in response to RANTES were cross-desensitized by prior exposure of the double transfectant cells to IL-8 (Table IV), although to a lower extent than the wild type CCR1 (Table I). Both CXCR1- and CXCR2-mediated Ca²⁺ mobilization in whole cells and GTPase activity in membranes were cross-desensitized upon activation of S3 and CCR1 by RANTES (Table IV, data not shown).

View this table: [in this window] [in a new window]   Table IV Cross-desensitization of S3 and CCR1-mediated Ca2+ mobilization by CXCR1 and CXCR2 RBL-2H3 cells (5 × 106 cells/assay) expressing S3-CXCR1, S3-CXCR2, CCR1-CXCR1, and CCR1-CXCR2 were loaded with Indo-1 and stimulated with either RANTES (10 nM) or IL-8 (10 nM). Cells were rechallenged 3 min later with a second dose of the indicated ligand and peak intracellular Ca2+ mobilization was determined. Data are the means ± S.E. of three different experiments.

CCR1 and CCR1-mediated PLC₃ Phosphorylation-- PLC₃ has been shown to be the only PLC isozyme expressed in RBL cells (21, 22). Whether CCR1 activation resulted in PLC₃ phosphorylation was studied. As shown in Fig. 11A, both CCR1 and CCR1 mediated RANTES-induced phosphorylation of PLC₃ to an extent similar (~2-fold over basal) (lanes 2 and 3) to that of CXCR1, CXCR2, and 331T (19). CCR1 induced phosphorylation of PLC₃ to an extent similar to that of cpt-cAMP (~2-fold over basal) (Fig. 11B, lanes 2 and 3) but lower than that of PMA (~3-fold over basal) (lane 4).

View larger version (31K): [in this window] [in a new window]   Fig. 11.   CCR1- and CCR1-mediated PLC3 phosphorylation. A, RBL-2H3 cells expressing CCR1 or CCR1 were 32P-labeled and stimulated for 5 min with RANTES (100 nM). Cells were lysed, immunoprecipitated with anti-PLC3 antibody, and analyzed by SDS-PAGE and autoradiography. B, 32P-labeled CCR1-expressing cells were stimulated for 5 min with RANTES (100 nM), cpt-cAMP (1 mM), or PMA (100 nM) and PLC3 phosphorylation was determined as described above. The results are from a representative experiment that was repeated three times.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemokines are inflammatory mediators of the chemotactic and cytotoxic functions of a large variety of cells including neutrophils, monocytes, eosiniphils, basophils, and lymphocytes. Most chemokines activate more than one receptor on leukocytes. This redundancy in receptor activation has hampered the investigation of their mechanisms of regulation. In this study, the CCR1 receptor, which binds RANTES, MIP-1, MCP-2, and MCP-3 with high affinity (1-3), was stably expressed in the leukocyte-like RBL-2H3 cell line. The data presented herein demonstrate that CCR1-mediated responses to RANTES, MIP-1, and MCP-2 are regulated via receptor phosphorylation-dependent and -independent mechanisms. First, prior exposure of cells expressing CCR1 to RANTES, MIP-1, MCP-2, or PMA, which causes phosphorylation of the receptor (Fig. 2), inhibited both Ca²⁺ mobilization in whole cells and GTPase activity in membranes. Second, stimulation with IL-8 of either CXCR1 (CXCR1-CCR1 cells) or CXCR2 (CXCR2-CCR1 cells), which results in CCR1 cross-phosphorylation, desensitized RANTES- and MIP-1-mediated GTPase activity and Ca²⁺ mobilization (Fig. 5 and Table I). Third, alanine substitution of specific serine and threonine residues as well as truncation of the cytoplasmic tail of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but not desensitization of receptor-mediated Ca²⁺ mobilization (Figs. 8 and 9, Table II).

The cAMP analog, cpt-cAMP, which caused phosphorylation of PLC₃ but not CCR1, inhibited Ca²⁺ mobilization to RANTES, MIP-1, and MCP-2 (Figs. 2 and 11). MIP-1 induced dose- and time-dependent increases in intracellular cAMP levels in the human megakaryocytic leukemia cell line M07e (23). In addition, RANTES- and MIP-1-mediated lymphocyte uropod formation and adhesion receptor redistribution were inhibited by the cAMP-dependent protein kinase inhibitor H-89, suggesting a role for PKA as a downstream regulator of CCR1 (24).

All three CC chemokines tested herein homologously desensitized by >90% CCR1-mediated Ca²⁺ mobilization to a second dose of the same chemokine, and cross-desensitized Ca²⁺ response to each other (Fig. 2 and Table I). RANTES and MIP-1 cross-desensitized by >90% responses to a second dose of either chemokine, whereas MCP-2 blocked the response to both RANTES and MIP-1 by ~50%. Since MCP-2 mediated ~50% of the Ca²⁺ response elicited by RANTES and MIP-1, its lower rate of cross-desensitization may be due to its character as a partial agonist (4). Neote et al. (4) reported that, in human kidney 293 cells expressing CCR1, pretreatment of the cells with a first dose of MIP-1 abolished Ca²⁺ mobilization to a second dose of either MIP-1 or RANTES, whereas RANTES pretreatment only desensitized the response to a second dose of RANTES. The contrast between those results and the ones obtained in this work may indicate differences in the cell types in which the experiments were conducted. Several chemokine receptors, including CCR1, have been shown to couple to different G proteins to transduce signals, depending on the cell type in which they are being expressed (25).

Chemoattractant class-desensitization is a form of cross-inhibition of cellular responses (i.e. G protein turnover, PI hydrolysis, Ca²⁺ mobilization) as was demonstrated among a particular group of chemoattractant receptors (i.e. fMLP, C5a, and IL-8 but not platelet-activating factor and leukotriene B₄) (16, 26, 27). Whether "classes" of chemokine receptors cross-desensitize cellular responses to each other remained unclear. It was previously demonstrated that CXCR1 cross-desensitized responses to CXCR2 but not vice versa (19). In the present study, in cells co-expressing CCR1 and either CXCR1 or CXCR2, IL-8 stimulation inhibited RANTES- and MIP-1-mediated Ca²⁺ mobilization and GTPase activity (Table I and Fig. 5), suggesting that CXC chemokine receptors can down-regulate the cellular responses of a CC chemokine receptor. In addition, the results indicate that CCR1 cross-desensitization occurred at two levels: receptor/G protein uncoupling via receptor phosphorylation and inhibition of the activation of the downstream effector, PLC₃. This contention is based in the following observations. First, activation of either CXCR1, CXCR2 or 331T cross-phosphorylated CCR1 (Fig. 6) and inhibited CCR1-mediated GTPase activity and Ca²⁺ mobilization (Fig. 5 and Table I). Second, despite the resistance of S3 and CCR1 to cross-phosphorylation, their Ca²⁺ mobilization was blocked by activation of either CXCR1 or CXCR2 (Table III), although to a lesser extent than CCR1 (Table IV).

An unexpected finding is that CCR1 failed to cross-phosphorylate and cross-desensitize responses to CXCR1 (Fig. 6 and Table I). Previous studies in neutrophils and transfected RBL-2H3 cells have shown that responses to both CXCR1 and CXCR2, including GTPase activity and Ca²⁺ mobilization, are cross-desensitized by fMLP receptor and C5a receptor (19, 26-28). It was also shown that response to CXCR1 was resistant to cross-regulation by CXCR2 (19). However, truncation of the cytoplasmic tail of CXCR2 (331T), which prolongs its signaling and increases its resistance to internalization led to cross-regulation of CXCR1 (19, 29, 30). Thus, the resistance of CXCR1 to cross-desensitization by CCR1 may also be due to the strength of the CCR1-mediated signal, which may not be sufficient to trigger the cross-desensitization mechanism required for CXCR1, although it is sufficient to cross-desensitize CXCR2. Indeed, S3 and CCR1, which generated greater signals and were more resistant to internalization than CCR1 (3-5% (S3 and CCR1) versus ~90% (CCR1) after 60 min), cross-phosphorylated and cross-desensitized CXCR1 (Fig. 10, Table IV).

Of interest is that CCR1 cross-desensitized Ca²⁺ responses to CXCR2 as well as the phosphorylation-resistant mutant of CXCR2, 331T, to the same extent (Table I). These results indicate that cross-desensitization of Ca²⁺ mobilization among CCR1 and CXCR2 is independent of receptor phosphorylation and further suggest the importance of downstream effector(s) in receptor-mediated cross-desensitization. The downstream effector(s) involved in cross-desensitization of Ca²⁺ mobilization is not known. However, several studies have indicated that phosphorylation of PLC upon receptor activation may result in a decrease of PLC-mediated inositol trisphosphate production and, thus, inhibition of intracellular Ca²⁺ mobilization (21, 22, 30, 31). Indeed, CCR1 as well as CXCR1 and CXCR2 induced PLC₃ phosphorylation upon activation (Fig. 11). Nonetheless, phosphorylation of PLC₃ cannot of itself explain downstream cross-desensitization since, despite mediating PLC phosphorylation to the same extent (~2-fold over basal), both CCR1 and CXCR2 failed to cross-desensitize responses to CXCR1. Thus, an additional process must be involved. Since the only PLC isozyme expressed in RBL cells is PLC₃ (21), this result may indicate that cross-desensitization requires modification of an additional signaling component needed to activate PLC₃ and that CXCR1 versus CCR1 and CXCR2 use different pathways. Supporting that contention is the report that, in addition to phosphorylation of PLC, modification of either G proteins or G protein-related proteins such as RGS (regulators of G protein signaling) may also required for the regulation of PLC signaling (32).

In summary, these data demonstrate that CCR1-mediated responses to RANTES, MIP-1, and MCP-2 are regulated via multiple mechanisms of desensitization including homologous (presumably via a GRK-dependent mechanism), heterologous (via second messenger activated kinases), and class desensitization (inhibition of PLC activation) by CXC chemokines. In addition, they demonstrate that CCR1 and CXCR2 cross-desensitize each other at two levels: receptor/G protein coupling and modification of downstream effector. CXCR1 was resistant to cross-desensitization by CCR1 but not by its phosphorylation- and internalization-resistant mutants S3 and CCR1. This suggests a role for signal strength in chemokine receptor cross-regulation, which is regulated by phosphorylation of domains in the receptor cytoplasmic tail. Given the multiplicity of chemokine receptor for identical ligands, it is likely that the evolution of receptors with similar ligand specificity but different signal lengths based on cytoplasmic tail phosphorylation sites plays an important role in immunoregulation. Thus, the ability of such classes of CC and CXC chemokine receptors to selectively cross-regulate each other at multiple levels may be physiologically relevant in controlling immune response.

	ACKNOWLEDGEMENTS

We are grateful to Drs. Timothy N. C. Wells and Ann Richmond for the gift of cDNAs encoding the CCR1 and 331T mutant, respectively

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.), AI-43184 (to B. H.), and DE-03738 (to R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, Box 3680, Durham, NC 27710. Tel.: 919-684-5332; Fax: 919-684-4390; E-mail: richa021@mc.duke.edu.

	ABBREVIATIONS

The abbreviations used are: RANTES, regulated upon activation normal T expressed and secreted; MIP-1, macrophage inflammatory protein-1; MCP-2, monocyte chemotactic protein-2; fMLP, formylmethionylleucylphenylalanine; C5a, complement cleavage product; CCR1, RANTES receptor; IL-8, interleukin-8; CXCR1, IL-8 receptor A; CXCR2, IL-8 receptor B; PMA, phorbol 12-myristate 13-acetate; G protein, GTP-regulatory protein; PLC, phospholipase C; HA, hemagglutinin; cpt-cAMP, 8-(4-chlorophenylthio)-adenosine 3'-cyclic monophosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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