Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2.

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-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) 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 Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, 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 Ca(2+) mobilization) which is inversely correlated 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.

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 B 4 , 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. 1 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 3Ј-cyclic monophosphate.  I-RANTES and 125 I-IL-8 were obtained from Amersham Pharmacia Biotech. RANTES, IL-8 (monocytederived), 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 PLC␤ 3 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 3Ј-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.

EXPERIMENTAL PROCEDURES
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 5Ј oligonucleotide corresponding to the epitope-tagged CCR1 (YPYDVPDYA) was used with a 3Ј 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 ϫ 10 7 cells) were transfected by electroporation with pcDNA3 containing the receptor cDNAs (20 g), 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 ϫ 10 6 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 l) 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 l of radioimmune precipitation buffer, and bound radioactivity was counted (18,19). Nonspecific radioactivity bound was determined in the presence of 1 M unlabeled ligand. For internalization, cells were incubated with different ligands for 0 -60 min. Then cells were washed with phosphate-buffered saline, and 125 I-RANTES or 125 I-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 Ci/ml [ 3 H]inositol. The generation of inositol phosphates was determined as reported (17). For calcium mobilization, cells (3 ϫ 10 6 ) were removed, washed with HEPES-buffered saline, and loaded with 1 M Indo I-AM in the presence of 1 M 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 PLC␤ 3 -Phosphorylation of receptors or PLC␤ 3 was performed as described previously (17)(18)(19). RBL (5 ϫ 10 6 ) expressing the receptors were incubated with [ 32 P]orthophosphate (150 Ci/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 NH 2 terminus of CXCR1 or CXCR2; the HA epitope tag of CCR1 or the PLC␤ 3 , 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 g of membrane preparations was carried out as described previously (18,19).

Expression and Characterization of CCR1 in RBL-2H3
Cells-Competition binding assays using 125 I-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 Ϯ 2 nM and B max of 7354 Ϯ 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 Ϯ 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 EC 50 values for RANTES were 2.17 Ϯ 0.25 nM and 3.3 Ϯ 0.65 nM for PI hydrolysis and exocytosis, respectively. CCR1 mediated comparable peak intracellular Ca 2ϩ mobilization in response to both RANTES and MIP-1␣ (Fig. 1C). MCP-2-induced Ca 2ϩ mobilization was ϳ50% 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, Ca 2ϩ mobilization, and secretion (data not shown).
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.
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 125 I-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 Ͼ 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 Ca 2ϩ 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).
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 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.
indicate that the phosphorylation sites for CCR1 are located in the cluster of serine and threonine which comprises amino acids 340 -346. Ca 2ϩ 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 (ϳ67%, ϳ59%, and 61%, respectively) were more resistant to desensitization than S1 and CCR1 (ϳ82% and 86%, respectively).
S3-and ⌬CCR1-mediated Ca 2ϩ 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 Ca 2ϩ 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).

DISCUSSION
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 phos-  8 (B and D) binding. The values are presented as percentage of total, which is defined as the total amount of 125 I-ligand bound to control (untreated) cells. The experiment was repeated four times with similar results.

TABLE II
Amino acid sequences of the carboxyl-terminal tail of the wild type CCR1 and the serine and threonine residues either replaced with alanine or truncated in each mutants Bold serine and threonine residues are potential phosphorylation sites in the wild type CCR1.

308
355 phorylation-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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ mobilization ( Figs. 8 and 9, Table II). The cAMP analog, cpt-cAMP, which caused phosphorylation of PLC␤ 3 but not CCR1, inhibited Ca 2ϩ mobilization to RAN-TES, 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). 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, 32 P-labeled RBL-2H3 cells (5 ϫ 10 6 ) 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.
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 ϫ 10 6 ) were loaded with Indo-1 and RANTES (10 nM)-stimulated Ca 2ϩ 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.
All three CC chemokines tested herein homologously desensitized by Ͼ90% CCR1-mediated Ca 2ϩ mobilization to a second dose of the same chemokine, and cross-desensitized Ca 2ϩ response to each other ( Fig. 2 and Table I). RANTES and MIP-1␣ cross-desensitized by Ͼ90% responses to a second dose of either chemokine, whereas MCP-2 blocked the response to both RAN-TES and MIP-1␣ by ϳ50%. Since MCP-2 mediated ϳ50% of the Ca 2ϩ 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 Ca 2ϩ 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, Ca 2ϩ 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 B 4 ) (16,26,27). Whether "classes" of chemokine receptors crossdesensitize 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 Ca 2ϩ 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, PLC␤ 3 . 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 Ca 2ϩ mobilization ( Fig. 5 and Table I). Second, despite the resistance of S3 and ⌬CCR1 to cross-phosphorylation, their Ca 2ϩ 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 Ca 2ϩ 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 crossregulation 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 ϳ90% (CCR1) after 60 min), cross-phosphorylated and cross-desensitized CXCR1 (Fig. 10, Table IV).
Of interest is that CCR1 cross-desensitized Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ mobilization (21,22,30,31). Indeed, CCR1 as well as CXCR1 and CXCR2 induced PLC␤ 3 phosphorylation upon activation (Fig. 11). Nonetheless, phosphorylation of PLC␤ 3 cannot of itself explain downstream cross-desensitization since, despite mediating PLC␤ phosphorylation to the same extent (ϳ2-fold over basal),  11. CCR1-and ⌬CCR1-mediated PLC␤ 3 phosphorylation. A, RBL-2H3 cells expressing CCR1 or ⌬CCR1 were 32 P-labeled and stimulated for 5 min with RANTES (100 nM). Cells were lysed, immunoprecipitated with anti-PLC␤ 3 antibody, and analyzed by SDS-PAGE and autoradiography. B, 32 P-labeled CCR1-expressing cells were stimulated for 5 min with RANTES (100 nM), cpt-cAMP (1 mM), or PMA (100 nM) and PLC␤ 3 phosphorylation was determined as described above. The results are from a representative experiment that was repeated three times. 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 PLC␤ 3 (21), this result may indicate that cross-desensitization requires modification of an additional signaling component needed to activate PLC␤ 3 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.