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J. Biol. Chem., Vol. 279, Issue 31, 32524-32533, July 30, 2004

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The CXC Chemokine Receptor Encoded by Herpesvirus saimiri, ECRF3, Shows Ligand-regulated Signaling through G_i, G_q, and G_12/13 Proteins but Constitutive Signaling Only through G_i and G_12/13 Proteins^*

Mette M. Rosenkilde, Katherine A. McLean, Peter J. Holst, and Thue W. Schwartz

From the Laboratory for Molecular Pharmacology, Department of Pharmacology, the Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark

Received for publication, December 8, 2003 , and in revised form, May 14, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Open reading frame 74 (ORF74) of many ₂ -herpesviruses encodes a CXC chemokine receptor. The molecular pharmacological profile of ORF74 from herpesvirus saimiri, ECRF3, is characterized here and compared with that of the well known ORF74 from human herpesvirus 8 (HHV8). The ECRF3 receptor bound the so-called ELR (Glu-Leu-Arg) CXC chemokines ¹²⁵I-CXCL1/GRO, ¹²⁵I-CXCL6/GCP-2, and ¹²⁵I-CXCL8/interleukin-8 with high affinity; but in contrast to ORF74 from HHV8, it did not bind the non-ELR CXC chemokine ¹²⁵I-CXCL10/IP10. Interestingly, the B_max value for CXCL6/GCP-2 was 3-fold higher than the capacity for maximal binding of CXCL1/GRO to ECRF3 and 85-fold higher than that of CXCL8/interleukin-8, despite similar affinities. Like ORF74 from HHV8, ECRF3 activated a broad range of pathways (G_q, G_i, and G_12/13 as well as the cAMP response element-binding protein, NF-B, NFAT, and serum response element transcription factors) in a ligand-regulated manner, with CXCL6/GCP-2 being the most potent and efficacious agonist. ECRF3 signaled constitutively through G_i and G_12/13, but surprisingly not through G_q. At the level of transcription factor activation, the serum response element was activated constitutively by ECRF3, whereas cAMP response element-binding protein, NFAT, and NF-B were only ligand-regulated. The maximal signaling capacities were similar for the two receptors; however, the ligand-regulated signaling was responsible for the major part of the total ECRF3 signaling and only for a minor part of the total HHV8 ORF74 signaling. The activation pattern of ECRF3 with constitutive activation of some (but not all) of the employed pathways has not been seen before in endogenous or virus-encoded chemokine receptors. The results suggest that the unique ligand selectivity of ECRF3 among ORF74 receptors could reflect differences in the cellular tropism of the ₂-herpesviruses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemokine receptors belong to the family of rhodopsin-like seven-transmembrane (7TM)¹ -helix receptors (1). Upon activation, the 7TM receptors mainly transduce signals to G-proteins that are divided into four main classes: G_i/o, G_q, G_s, and G_12/13. Apart from the odorant receptors, the majority of 7TM receptors (43%) activate G_i (2). Most 7TM receptors interact selectively with one G-protein; however, 11% of 7TM receptors signal through more than one G-protein (2). Signal transduction of chemokine receptors is mediated mainly by pertussis toxin (PTx)-sensitive calcium release and chemotaxis pointing at G_i as the principal pathway. A variety of signal transduction pathways initiated by G_i as well as by the -subunits have been described for chemokine receptors (reviewed in Ref. 3). Chemokines are divided in two major groups, CC and CXC chemokines, based on the presence or absence of a residue between the first two of four conserved cysteines. The CXC chemokines are divided into two groups based on the presence or absence of an ELR (Glu-Leu-Arg) motif just prior to the first cysteine (1).

The chemokine system has been subject to molecular piracy by certain viruses. Thus, some pox- and herpesviruses encode chemokines and/or chemokine receptors in their genomes (4). The virus-encoded chemokine receptors are structurally rather divergent and have in general evolved very different functional properties compared with their human homologs, indicating remarkable evolutionary pressure. The open reading frame 74 (ORF74) receptors constitute one cluster of synthetic genes (all encoded by ORF74) and are shared by many members of the rhadinovirus lineage (₂-herpesviruses) (Fig. 1) (5). Thus, ORF74 receptors have been cloned from murid -herpesvirus 68 (ORF74-MHV68), equine herpesvirus 2, human herpesvirus 8 (ORF74-HHV8), herpesvirus saimiri (ECRF3 or ORF74-HVS), and the closely related atheles herpesvirus. The ORF74 family shows the highest structural homology to mammalian CXC chemokine receptor (CXCR) 2, and the functional characterization of ORF74-HHV8 (6–8), ECRF3 (9), and ORF74-MHV68 (10) as being CXC chemokine receptors supports the structural homology.

View larger version (54K): [in this window] [in a new window]   FIG. 1. Dendrogram of herpesvirus-encoded chemokine receptors. The phylogenetic tree was constructed using ClustalW Version 1.5 alignment of the whole sequence, followed by analysis using the Distance program of the GCG Wisconsin package (kindly provided by Francois Talabot, Ares-Serono). Constitutively active receptors are marked in red. Solid lines indicate a more peripheral cluster association. CMV, cytomegalovirus; EHV2, equine herpesvirus 2; AtHV, atheles herpesvirus.

The general knowledge of 7TM receptor signal transduction mechanisms and knowledge of the properties of virus-encoded chemokine receptors have developed considerably since the first description of ECRF3 from HVS in 1993 (9). In this study, we characterized ECRF3 on this basis and surprisingly found that it has a very interesting molecular pharmacological phenotype in being constitutively active through G_i and G_12/13 (the latter shown by studying the serum response element (SRE) transcription factor in the presence of pathway-specific inhibitors), whereas it activates a broader spectrum of signaling pathways in a ligand-regulated manner, with CXCL6/GCP-2 being the most potent agonist.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The human chemokines were purchased from Peprotech (CXCL6/GCP-2, CXCL1/GRO, CXCL12/stromal cell-derived factor 1, and CXCL10/IP10); kindly provided by Dr. Timothy N. C. Wells (Serono Pharmaceutical Research Group, Ares-Serono, Geneva, Switzerland) (vCCL2/vMIP-II); or made in-house by Dr. Thomas P. Boesen through Escherichia coli expression, purification, and refolding (CXCL8/IL-8). The ECRF3 receptor was kindly provided by Dr. John Nicholas (Johns Hopkins Oncology Center, Baltimore, MD). ORF74 from HHV8 (GenBank^TM/EBI accession number U24275) was cloned from a Kaposi's sarcoma skin lesion biopsy taken from a human immunodeficiency virus 1-infected patient (21). LipofectAMINE^TM 2000 reagent and Opti-MEM I (catalog no. 51985-026) were purchased from Invitrogen. LucLite (lyophilized substrate solution) was from PerkinElmer Life Sciences. Monoiodinated ¹²⁵I-CXCL8/IL-8 and ¹²⁵I-CXCL1/GRO, myo-[³H]inositol (PT6-271), [³H]adenine (TRK311), and Bolton-Hunter reagent for iodination of CXCL6/GCP-2 and CXCL10/IP10 were from Amersham Biosciences (Uppsala, Sweden). AG 1-X8 anion-exchange resin (for phosphatidylinositol (PI) turnover) and AG-50W-X4 resin (for cAMP assay) were from Bio-Rad. Alumina, imidazole, 3-isobutyl-1-methylxanthine, and PTx (for cAMP assay) were from Sigma. U-73122, Y-27632, and C3 exoenzyme from Clostridium botulinum were from Calbiochem and Merck Biosciences (Nottingham, United Kingdom).

Iodination of CXCL6/GCP-2 and CXCL10/IP10—The Bolton-Hunter reagent was dried under a gentle stream of nitrogen for 30–60 min. 5–10 µg of chemokine was incubated on ice with 1.5 mCi of Bolton-Hunter reagent in a total volume of 50 µl of 0.1 mM borate buffer (pH 8.5) for 1 h. The reaction was terminated by addition of 0.25 ml of H₂O supplemented with 0.1% (v/v) trifluoroacetic acid. The labeled chemokines were purified by reverse-phase high pressure liquid chromatography.

Transfections and Tissue Culture—COS-7 cells were grown at 10% CO₂ and 37 °C in Dulbecco's modified Eagle's medium with Glutamax (catalog no. 21885-025, Invitrogen) adjusted with 10% fetal bovine serum, 180 units/ml penicillin, and 45 µg/ml streptomycin. HEK293 cells were grown in Dulbecco's modified Eagle's medium adjusted to contain 4500 mg/liter glucose with 10% fetal bovine serum, 180 units/ml penicillin, and 45 µg/ml streptomycin at 10% CO₂ and 37 °C. The HEK293 cell medium was modified to contain heat-inactivated fetal bovine serum and no penicillin and streptomycin during luciferase-based assays. Transfection of the COS-7 cells was performed by the calcium phosphate precipitation method (8) for the competition binding and PI turnover experiments and by the cationic lipid reagent method with LipofectAMINE^TM 2000 reagent in serum-free Opti-MEM I according to the manufacturer's recommendations for the luciferase-based transcription factor experiments. HEK293 cells were transfected by the calcium phosphate precipitation method (8) for the adenylate cyclase inhibition experiments and with LipofectAMINE^TM 2000 reagent in serum-free Opti-MEM I according to the manufacturer's recommendations for the luciferase-based transcription factor experiments.

Binding Experiments—COS-7 cells were transferred to culture plates 1 day after transfection. The number of cells seeded per well was determined by the apparent expression efficiency of the receptors, with the goal of obtaining 5–10% specific binding of the added radioactive ligand. 3–5 x 10⁵ cells/well were used to test specific binding. 2 days after transfection, cells were assayed by competition binding for 3 h at 4 °C using 12 pM ¹²⁵I-CXCL8/IL-8, ¹²⁵I-CXCL1/GRO, ¹²⁵I-CXCL6/GCP-2, or ¹²⁵I-CXCL10/IP10 plus unlabeled ligand in 0.5 ml of 50 mM Hepes (pH 7.4) supplemented with 1 mM CaCl₂, 5 mM MgCl₂, and 0.5% (w/v) bovine serum albumin. After incubation, cells were washed quickly four times with 4 °C binding buffer supplemented with 0.5 M NaCl. Nonspecific binding was determined as the binding in the presence of 0.1 µM unlabeled chemokine. Determinations were made in duplicates.

Phosphatidylinositol Assay (PI Turnover)—1 day after transfection, COS-7 cells (2.5 x 10⁴/well) were incubated for 24 h with 5 µCi/ml myo-[³H]inositol in 0.3 ml/well growth medium. Cells were washed twice with 20 mM Hepes (pH 7.4), 140 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM glucose, and 0.05% (w/v) bovine serum albumin and were incubated in 0.3 ml of the same buffer supplemented with 10 mM LiCl at 37 °C for 90 min in the presence or absence of chemokines. PTx (100 ng/ml) were added 18 h prior to the experiment. Cells were extracted by addition of 1 ml/well 10 mM formic acid, followed by a 30-min incubation on ice. The generated [³H]inositol phosphates were purified on AG 1-X8 anion-exchange resin (22). Determinations were made in duplicates.

Adenylate Cyclase Inhibition Assay (cAMP Assay)—1 day after transfection, HEK293 cells (2.5 x 10⁴/well) were incubated for 24 h with 2 µCi/ml [³H]adenine in 0.5 ml/well growth medium. Cells were washed twice with 25 mM Hepes (pH 7.2), 0.75 mM NaH₂PO₄, 140 mM NaCl, and 0.05% (w/v) bovine serum albumin, and 0.5 ml of the same buffer supplemented with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) was added together with the adenylate cyclase activator forskolin (15 µM) to the cells. Ligands were then added, and the reaction was terminated after a 15-min incubation at 37 °C. After incubation, the cells were placed on ice; the medium was removed; and the cells were lysed in 1 ml of 5% (w/v) trichloroacetic acid supplemented with 0.1 mM cAMP and 0.1 mM ATP for 30 min. The lysis mixtures were loaded onto Dowex columns, which were washed with 2 ml of water, placed on the top of alumina columns, and washed again with 10 ml of water. The alumina columns were eluted with 6 ml of 0.1 M imidazole into 15 ml of scintillation fluid (Highsafe III). Columns were reused up to 15 times. Dowex columns were regenerated by adding 10 ml of 2 N HCl followed by 10 ml of water; the alumina columns were regenerated by adding 2 ml of 1 M imidazole, 10 ml of 0.1 M imidazole, and finally 5 ml of water. The effect of 100 ng/ml PTx was tested by adding it to the cells 18 h before ligand addition. Determinations were made in triplicates.

Constitutive SRE, NFAT, and NF-B cis-Reporting and CREB trans-Reporting Luciferase Assays—Cells were seeded at 35,000 cells/well in culture plates 24 h prior to transfection and were transfected with the cis-reporter plasmid (pSRE-Luc, pNFAT-Luc, or pNF-B-Luc; 50 ng/well) and the receptor plasmid (0–50 ng/well). For the trans-reporting system, 50 ng/well trans-activator plasmid (pFR-Luc) was added together with 6 ng/well trans-reporter plasmid (pFA2-CREB) and receptor plasmids. 24 h after transfection, the cells were washed twice with Dulbecco's phosphate-buffered saline, and luminescence was measured in a microplate scintillation and luminescence counter (Top-counter, PerkinElmer Life Sciences) after a 10-min incubation in 100 µl of Dulbecco's phosphate-buffered saline together with 100 µl of LucLite substrate. All determinations were made in quadruplicates.

Ligand and Inhibitor Modification of Transcription Factor Activity— The cells were transfected with 10 ng/well receptor cDNA and the above-mentioned concentrations of reporter and activator plasmids. Chemokine ligands were added at concentrations ranging from 10^–11 to 10^–7 M for the dose-response curves. Alternatively, a constant concentration (100 nM) was added to the receptor gene dose experiments (see above). In both cases, as well as upon addition of the inhibitors PTx (100 ng/ml) and U-73122 (10 µM) to the CREB, NF-B, and NFAT transcription factors and of PTx (100 ng/ml), C3 exoenzyme (10 µg/ml), and Y-27623 (10 µM) to the SRE transcription factor, the chemokines/inhibitors were added immediately following transfection, and luminescence was measured 24 h post-transfection as described above. All determinations were made in quadruplicates.

Calculations—IC₅₀ and EC₅₀ values were determined by nonlinear regression, and B_max values were calculated using the GraphPad Prism Version 2 software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The CXC Chemokine Binding Profile of ECRF3 Is Limited Exclusively to the ELR Motif-containing CXC Chemokines— Homolog competition binding analysis was performed in transiently transfected COS-7 cells with four radioligands, including two previously described agonists for ECRF3, ¹²⁵I-CXCL1/GRO and ¹²⁵I-CXCL8/IL-8 (9), together with ¹²⁵I-CXCL10/IP10 and ¹²⁵I-CXCL6/GCP-2, which are full and partial inverse agonists for ORF74-HHV8, respectively. All three ELR CXC chemokines (CXCL1/GRO, CXCL8/IL-8, and CXCL6/GCP-2) bound specifically to ECRF3 with high and remarkably similar binding affinities (IC₅₀) of 0.25 ± 0.07, 0.46 ± 0.17, and 0.51 ± 0.21 nM, respectively (Fig. 2, A–C). These affinities resembled the corresponding affinities for ORF74-HHV8 (Fig. 2, A–C). The non-ELR CXC chemokine CXCL10/IP10 displayed no specific binding to ECRF3, in contrast to the high affinity for ORF74-HHV8 (Fig. 2D). Interestingly, large differences in the maximal binding capacities were assessed despite the high and similar binding affinities. Thus, B_max was 12 ± 2.5 fmol/10⁵ cells for ¹²⁵I-CXCL1/GRO, whereas it was only 0.4 ± 0.1 fmol/10⁵ cells for ¹²⁵I-CXCL8/IL-8. In contrast, ¹²⁵I-CXCL6/GCP-2 displayed a B_max of 34 ± 6.7 fmol/10⁵ cells, nearly three times higher compared with ¹²⁵I-CXCL1/GRO and 85 times higher compared with ¹²⁵I-CXCL8/IL-8. These B_max values were in striking contrast to the almost similar values for ¹²⁵I-labeled CXCL1/GRO, CXCL8/IL-8, and CXCL6/GCP-2 for binding to ORF74-HHV8 (33 ± 5.9, 39 ± 6.8, and 26 ± 9.4 fmol/10⁵ cells, respectively), obtained in parallel with the B_max values for ECRF3. A larger range of ELR as well as non-ELR CXC chemokines was analyzed by heterologous competition binding against ¹²⁵I-CXCL1/GRO (Table I). All three GRO peptides (CXCL1–3/GRO,,) bound with high affinities to ECRF3, whereas the apparent affinity of CXCL7/NAP-2 was only 155 nM. The non-ELR CXC chemokines CXCL12/stromal cell-derived factor 1 and CXCL10/IP10 displayed very low affinities for ECRF3 (1016 and 622 nM, respectively), in agreement with the absence of any specific binding for CXCL10/IP10. Previously, a selection of endogenous CC chemokines was tested (CCL3/MIP-1, CCL5/RANTES (regulated on activation normal T-cell expressed and secreted), CCL2/monocyte chemoattractant protein 1, and CCL4/MIP-1) and found not to interact with ECRF3 (9). Therefore, no further analysis was performed in our system with respect to endogenous CC chemokines. However, the HHV8-encoded CC chemokine vCCL2/vMIP-II was found to have a surprisingly high affinity (IC₅₀ = 4.1 nM) (Table I) compared with its affinity of 72 nM for ORF74-HHV8 (8). In summary, ECRF3 bound a large spectrum of CXC chemokines with affinities varying from 0.4 to 155 nM. The specific binding was, however, strictly limited to the ELR CXC chemokines (no binding of the non-ELR CXC chemokines), in contrast to the broad-spectrum ELR and non-ELR CXC chemokine binding properties of ORF74-HHV8 and ORF74-MHV68 (8, 10, 13).

View larger version (36K): [in this window] [in a new window]   FIG. 2. Homologous competition binding to ECRF3. Transiently transfected COS-7 cells were used for the displacement of 125I-CXCL1/GRO (A), 125I-CXCL8/IL-8 (B), 125I-CXCL6/GCP-2 (C), and 125I-CXCL10/IP10 (D) by the homologous unlabeled chemokine from ECRF3 (, , , and ) or from ORF74-HHV8 (, , , and ). The total binding of 125I-CXCL10/IP10 to control cells () is included in D due to the lack of specific CXCL10/IP10 binding to ECRF3. All experiments were done in parallel (n = 3–6).

View larger version (27K): [in this window] [in a new window]   FIG. 3. ECRF3 activates Gq in a ligand-regulated manner. Transiently transfected COS-7 cells were used for the PI turnover assays. Chemokine dose-response curves are shown for ECRF3: CXCL6/GCP-2 (), CXCL1/GRO (), CXCL8/IL-8 (), CXCL7/NAP-2 (), CXCL10/IP10 (), and vCCL2/vMIP-II () (n = 5–11).

View larger version (18K): [in this window] [in a new window]   FIG. 5. PTx does not inhibit the PLC activation of ECRF3. Transiently transfected COS-7 cells were used for the PI turnover assays. A, increasing doses of CXCL6/GCP-2 applied to ECRF3 in the absence () or presence () of 100 ng/ml PTx; B, increasing doses of CXCL1/GRO applied to ORF74-HHV8 in the absence () or presence () of 100 ng/ml PTx. All experiments were done in parallel (n = 3).

View larger version (22K): [in this window] [in a new window]   FIG. 4. ECRF3 is not constitutively active through the Gq pathway. Transiently transfected COS-7 cells were used for the PI turnover assays. Increasing amounts of receptor/vector cDNA varying from 2.5 to 20 µg/3 x 106 cells were used for the transfection. A, basal activities of ECRF3 (), ORF74-HHV8 (), or pcDNA3-transfected cells (vector; ); B, agonist (10–7 M)-stimulated basal activities of ECRF3 () and ORF74-HHV8 (): CXCL6/GCP-2 activation of ECRF3 () and CXCL1/GRO activation of ECRF3 () and ORF74-HHV8 (). Arrows indicate agonist stimulation of ECRF3 (black) and ORF74-HHV8 (gray). All experiments were done in parallel (n = 5).

ECRF3 Activates a Broad Range of Downstream Transcription Factors in a Ligand-regulated (but Not Constitutive) Manner—Many virus-encoded chemokine receptors have been shown to signal constitutively through a broad range of transcription factors (26–31). In a gene dose setup using luciferase-based reporter systems for CREB, NF-B, and NFAT (three transcription factors with a known dependence on G_q activation) and receptors transiently expressed in HEK293 cells (Fig. 6), none of the transcription factors were constitutively activated by ECRF3. In contrast, all three were activated by increasing doses of ORF74-HHV8 in parallel with ECRF3 (Fig. 6, A–C, for CREB, NF-B, and NFAT, respectively) until a plateau was reached. This plateau was probably due to limitations in the content of intracellular signaling mediators from the cell surface to nucleus. The PI turnover experiments are not dependent on the same, many steps in the signaling cascade (PLC2 is closer to the receptor) and did not reach this plateau (Fig. 4). Moreover, receptor expression was higher in the luciferase-based transcription factor assays due to a more efficient transfection procedure. Upon addition of CXCL6/GCP-2 to the ECRF3-expressing cells, all three pathways were activated with remarkable high and almost identical potencies (EC₅₀) of 0.90, 0.70, and 0.42 nM for CREB, NF-B, and NFAT activities, respectively (Fig. 6, D–F). CXCL6/GCP-2 did not activate these transcription factors in cells transfected with the empty expression vector (data not shown). The same scenario of ligand regulation and lack of constitutive activity for ECRF3 (but maintained for ORF74-HHV8) was observed in transiently transfected COS-7 cells (data not shown).

View larger version (45K): [in this window] [in a new window]   FIG. 6. Transcription factor activity of ECRF3 compared with ORF74-HHV8. HEK293 cells were transiently transfected with ECRF3 (A–F) or ORF74-HHV8 (A–C) together with the transcription activation reporter CREB (pFA2-CREB) and the trans-activator (pFR-Luc) (A and D), the NF-B reporter (pNF-B-Luc) (B and E), and the NFAT reporter (pNFAT-Luc) (C and F). A–C, test for constitutive activation of transcription factors CREB, NF-B, and NFAT, respectively, by increasing receptor cDNA concentrations from 0 to 50 ng/well for ECRF3 (), ORF74-HHV8 (), and the empty expression vector pcDNA3 (). The experiments for each transcription factor were done in parallel for the two receptors and the expression vector. D–F, ligand-regulated activation of the different transcription factors with dose-response curves for the full agonist CXCL6/GCP-2 () upon ECRF3-mediated activation of CREB (D), NF-B(E), and NFAT (F)(n = 3–10). RLU, relative light units.

View larger version (45K): [in this window] [in a new window]   FIG. 7. Similar regulation of transcription activation by ECRF3 and ORF74-HHV8. HEK293 cells were transiently transfected with ECRF3 (A–C) and ORF74-HHV8 (D–F) together with the transcription activation reporter CREB (pFA2-CREB) and the trans-activator (pFR-Luc) (A and D), the NF-B reporter (pNF-B-Luc) (B and E), and the NFAT reporter (pNFAT-Luc) (C and F). Shown is the effect of the Gi inhibitor PTx (100 ng/ml) and the PLC inhibitor U-73122 (10 µM), applied either alone (, PTx; , U-73122) or together (), on the CXCL6/GCP-2 dose-response curve for ECRF3 (A–C) or the constitutive activity of ORF74-HHV8 (D–F). The activities in the absence of inhibitors are indicated (). The effect of the inhibitors on vector-transfected cells are indicated ( and ). The experiments for each transcription factor were done in parallel (n = 3–10).

View larger version (21K): [in this window] [in a new window]   FIG. 8. Identification of agonists and inverse agonists for the ECRF3-mediated activation of Gi. Transiently transfected HEK293 cells were used for the Gi assays. A, dose-response curves for CXCL6/GCP-2 (), CXCL1/GRO (), and vCCL2/vMIP-II () applied to the ECRF3 receptor after stimulation of adenylate cyclase with 15 µM forskolin (n = 5–6). B, forskolin stimulation of ECRF3-transfected HEK293 cells (right) or vector-transfected cells (left). White bars, basal activity; gray bars, 15 µM forskolin treatment; black and hatched bars, 50 µM forskolin treatment in the absence and presence of 100 ng/ml PTx, respectively (n = 4).

View larger version (36K): [in this window] [in a new window]   FIG. 9. Constitutive activation of the transcription factor SRE by ECRF3 and ORF74-HHV8. A and B, COS-7 cells were transiently transfected with increasing concentrations of ECRF3 (), ORF74-HHV8 (), or the expression vector pcDNA3 () together with the transcription activation SRE-luciferase reporter (pSRE-Luc). The effect of 100 ng/ml PTx on the basal receptor activity of ECRF3 () and on the expression vector () is shown in A, and the effect of PTx on the basal activity of ORF74-HHV8 () is shown in B. C, shown are the CXCL6/GCP-2 dose-response curves for the ECRF3 receptor in the absence () or presence () of 100 mg/ml PTx. D, shown are the CXCL1/GRO dose-response curves for the ORF74-HHV8 receptor in the absence () or presence () of 100 ng/ml PTx. E, shown is the effect of the Rho inhibitor C3 exoenzyme from C. botulinum (C3-exo; 10 µg/ml) and the Rho kinase inhibitor Y-27632 (10 µM) on the basal (black bars) and 10 nM CXCL6/GCP-2-induced (gray bars) stimulation of ECRF3. F, shown is the effect of C3 exoenzyme (10 µg/ml) and Y-27632 (10 µM) on the basal (black bars) and 10 nM CXCL1/GRO-induced (gray bars) stimulation of ORF74-HHV8. The gene dose experiments (A and B), the dose-response experiments (C and D), and the inhibitor experiments (E and F) were performed pairwise in parallel for the two receptors (n = 3–4). RLU, relative light units.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ligand Repertoire for ECRF3 Compared with Other ORF74 Receptors—In the initial study of ECRF3, CXCL1/GRO was found to be the most potent agonist, followed by CXCL8/IL-8 and CXCL7/NAP-2 (9). In agreement with this, ORF74-HHV8 was later shown to recognize the GRO peptides (CXCL1–3) as agonists, with CXCL1/GRO being the most potent agonist (6, 8, 13). Also ORF74 from MHV68 responds to GRO-related ELR motif-containing chemokines since the murine chemokines MIP-2 and KC (homologs of CXCL1/GRO) in addition to LIX (homolog of CXCL6/GCP-2 and CXCL5/ENA78) act as agonists for this receptor (10). Our analysis of ECRF3 confirms that CXCL1/GRO indeed is a highly potent agonist; however, another ELR CXC chemokine, CXCL6/GCP-2 was the most potent and efficacious agonist for ECRF3, with a 13 times higher potency and a 2.2 times higher efficacy with respect to G_q activation compared with CXCL1/GRO. The reason for the recognition of CXCL6/GCP-2 as the most potent agonist for ECRF3 and as an inverse agonist for ORF74-HHV8 (13) remains to be explained. The ELR CXC chemokines are chemoattractants of neutrophils and are angiogenic, whereas the non-ELR CXC chemokines attract lymphocytes and are in general angiostatic (40–42). Two ELR CXC chemokines (CXCL8/IL-8 and CXCL6/GCP-2) bind to the structurally related CXCR1 and CXCR2 (43, 44), whereas the rest, e.g. the GRO peptides (CXCL1–3), are selective for CXCR2 (45, 46). A selective non-peptide antagonist (SB 225002) has been developed for CXCR2 (47), and chemokines have been shown to bind differently to the two receptors (48, 49) and to be expressed differently (50). Thus, CXCL1/GRO and CXCL6/GCP-2 (and CXCL8/IL-8) differ with respect to receptor recognition and physiological roles, which could explain the choice of CXCL6/GCP-2 as a full agonist for ECRF3. The lack of agonistic properties of CXCL8/IL-8 for ECRF3 expressed in COS-7 or HEK293 cells is in contrast to the initial agonistic properties for ECRF3 expressed in Xenopus laevis oocytes (9). However, we observed a very low B_max for CXCL8/IL-8 compared with the B_max values for CXCL6/GCP-2 and CXCL1/GRO, and it could be anticipated that the lack of agonistic properties in our system was due to this diminutive B_max value for CXCL8/IL-8.

In this study, we observed no specific binding of the non-ELR CXC chemokine ¹²⁵I-CXCL10/IP10 to ECRF3, and binding analysis with ¹²⁵I-CXCL1/GRO revealed a very low affinity for CXCL10/IP10. Thus, the binding and inverse agonistic properties of CXCL10/IP10 observed for ORF74-HHV8 (8, 13, 14, 51) and the antagonistic properties of murine and human IP10 (CXCL10) for ORF74-MHV68 (10) are not shared by ECRF3. This lack of CXCL10/IP10 binding (and lack of modulation and down-regulation of ECRF3 by CXCL10/IP10) is, however, explainable in light of the cellular tropism for HVS since HVS has tropism for T-cells, in contrast to the B-cell tropism of HHV8 and MHV68 (8, 51). CXCR3 is highly up-regulated and expressed abundantly in activated T-cells, and the CXCR3 ligands (e.g. CXCL10/IP10) are present in high concentrations in T-cell-rich areas (1, 52). Thus, an interaction of ECRF3 with CXCL10/IP10 and consequent inhibition would be potentially harmful for HVS.

vCCL2/vMIP-II encoded by HHV8 is an unnatural ligand for ECRF3; and during in vivo settings, this ligand-receptor pair will never meet due to the virus-host discrepancy. Nevertheless, vCCL2/vMIP-II serves important purposes as determined in studies of virus-encoded receptors due to the promiscuous binding to the majority of endogenous and viral CC, CXC, XC, and CX₃C chemokine receptors, in most cases as an antagonist (8, 14, 21, 33) and more seldom as an agonist (53). We employed vCCL2/vMIP-II as a prototype antagonist and observed inverse agonistic and antagonistic properties of this chemokine for ECRF3.

Constitutive and Non-constitutive Activities of ECRF3—Besides the broad-spectrum ligand binding and the exploitation of several signal transduction pathways, the majority of virus-encoded receptors are unique due to the high degree of constitutive activity they usually display through several pathways (Table II) (6, 8, 26, 30, 54–58), in contrast to the quiescent endogenous chemokine receptors. In general, high constitutive activity is not common for endogenous 7TM receptors (59, 60); however, some receptors display high constitutive activity, e.g. the ghrelin and histamine receptors (61, 62), whereas the majority display low to undetectable levels of constitutive activity (60). In this study, we have characterized signaling pathways in close proximity to the receptor (G_i and G_q activation and G_12/13 indirectly through SRE) and signaling mediators farther downstream (transcription factors CREB, NFAT, NF-B, and SRE). In contrast to the rest of the constitutively active virus-encoded receptors, we found, for ECRF3, selectivity in the constitutive activation of two of the three G-proteins investigated (G_i and G_12/13, but not G_q). The constitutive G_i activity was measured directly by looking at the inhibition of cAMP production in transiently transfected HEK293 cells, in which vCCL2/vMIP-II functioned as an inverse agonist and CXCL6/GCP-2 functioned as an agonist (Fig. 8A) and in which PTx reversed the constitutive adenylate cyclase inhibition by ECRF3 seen after forskolin treatment (Fig. 8B). We also measured the constitutive G_i activity indirectly from the SRE activity in the presence of pathway-specific inhibitors since SRE has been described as being dependent on G_i as well as on G_12/13 (32, 35, 36). The constitutive SRE activity was 4–5-fold lower for ECRF3 compared with ORF74-HHV8 (tested in parallel) (Figs. 9B and 10 and Table III). We applied the G_i inhibitor PTx and the Rho and Rho kinase inhibitors C. botulinum C3 exoenzyme and Y-27632, respectively (Rho and Rho kinase are downstream mediators of G_12/13 activation) (32, 35, 36), and observed partial inhibition of the constitutive as well as ligand-regulated activities of all three inhibitors, indicating a contribution of G_i as well as G_12/13 to the SRE activation of these two receptors. The G_i contribution to the basal and ligand-regulated activities of ECRF3 was 50% as judged from the influence of the inhibitors (47–55% inhibition by PTx concomitant with 34–67% inhibition by Y-27632 and C3 exoenzyme of the constitutive and ligand-regulated activities of ECRF3) (Fig. 9, A, C, and E). For ORF74-HHV8, the G_i pathway was most important for the basal SRE activity (60–65% inhibition by PTx concomitant with 31–46% inhibition by Y-27632 and C3 exoenzyme, respectively) (Fig. 9, B and F), whereas G_12/13 was most important for the ligand-regulated signaling (small effect of PTx and 65–71% inhibition by C3 exoenzyme and Y-27632) (Fig. 9, D and F). Thus, using these inhibitors, it became clear that the G_i activity (from SRE activation) was 6–7-fold lower for ECRF3 than for ORF74-HHV8, whereas the G_12/13 activity was 3–4-fold lower for ECRF3 than for ORF74-HHV8 (Fig. 10 and Table III). G_12/13 activation by ORF74-HHV8 is consistent with previously published data (31).

View larger version (25K): [in this window] [in a new window]   FIG. 10. Quantitative comparison of ECRF3 and ORF74-HHV8 signaling properties. A, the ligand-regulated (gray bars) and constitutive (black bars) activities of ECRF3 and ORF74-HHV8 (expressed in parallel in transiently transfected COS-7 cells) are shown here for the three different G-proteins (Gq, Gi, and G12/13) activated by both receptors. 100% equals the total activity (ligand-regulated + constitutive) of ORF74-HHV8 for each pathway. B, the signaling pattern with respect to SRE activation is also provided since the analysis of this transcription factor provided the data for the Gi and G12/13 activities (by addition of pathway-specific inhibitors to SRE activity). The constitutive ORF74/ECRF3 activity ratio for each pathway is given by the height of the black bars; the ligand-regulated ORF74-HHV8/ECRF3 activity ratio is given by the height of the gray bars; and the ligand-regulated/constitutive activity ratio for each receptor in each pathway is given by the height of the gray versus black bars. (Table III summarizes these ratios.)

Ligand-regulated Activities of ECRF3—Despite the lack of constitutive activity for G_q, CREB, NF-B, and NFAT, we observed a brilliant ligand-regulated signaling through all pathways for ECRF3. Thus, CXCL6/GCP-2 induced a 5.4-fold increase above the basal non-constitutive G_q activity of ECRF3, resulting in a maximal stimulation similar to the maximal CXCL1/GRO-mediated stimulation of ORF74-HHV8 (Fig. 4), tested in parallel for the two receptors. The PI turnover was not inhibited by the presence of PTx, indicating that the PLC activity is indeed a result of G_q activity in COS-7 cells and not mediated by the -subunits released from activated G_i. In fact, an increase in efficacy for both receptors (as well as in the constitutive activity of ORF74-HHV8) occurred in the presence of PTx (Fig. 5B), indicating that inhibition of G_i potentiates PLC activation. This phenomenon has been observed before for ORF74-HHV8 expressed in endothelial cells (27) and is explainable by the law of mass action since we found that ECRF3 couples to several G-proteins, (G_i, G_q, and G_12/13); the same has been shown for ORF74-HHV8 (6, 27, 29, 31, 63). A receptor in the active state couples only to one G-protein at a given time (64), and elimination of active G_i by PTx could raise the probability for an interaction with another G-protein and consequently result in an increase in PI turnover, as we observed for both receptors.

The CREB, NF-B, NFAT, and SRE transcription factors were activated 2–5-fold above the basal activity of ECRF3 by CXCL6/GCP-2 (Figs. 6 and 9). SRE activation has never been characterized before for ORF74-HHV8. We observed a high constitutive SRE activity for ORF74-HHV8 that was increased further by CXCL1/GRO, whereas the constitutive ECRF3 activity was increased further by CXCL6/GCP (Fig. 9, C–F).

Quantitative Comparison of ECRF3 and ORF74-HHV8 Signaling Properties—Not only did the two receptors express very similarly as determined from the B_max values for their full agonists (¹²⁵I-CXCL6/GCP-2 binding to ECRF3 with a B_max of 34 ± 6.7 fmol/10⁵ cells and ¹²⁵I-CXCL1/GRO binding to ORF74-HHV8 with a B_max of 33 ± 5.9 fmol/10⁵ cells), the total signaling capacities or efficiencies of ECRF3 and ORF74-HHV8 were also very similar. Thus, both in terms of G_q (Fig. 4) and G_i and G_12/13 coupling, based on SRE activation in the absence/presence of inhibitors (Fig. 9), we observed a total stimulation in the same range for these two receptors (Fig. 10, full bars; and Table III). However, whereas ORF74-HHV8 displayed very high constitutive signaling (between 50 and 80% of the maximal signaling capacity) (Fig. 10, black bars; and Table III), in all signaling pathways that it had the ability to signal through, ECRF3 displayed clear constitutive activity (14–20% of the maximal signaling capacity) only through the G_i, G_12/13, and SRE pathways (Fig. 10, black bars; and Table III). In contrast to ORF74-HHV8, no sign of constitutive signaling was observed for ECRF3 through G_q or through the other tested transcription factors, despite the fact that the full agonist for ECRF3 stimulated this receptor to the same maximal signaling capacity as that observed for ORF74-HHV8 (Fig. 10 and Table III). Since the maximal signaling capacities in the various pathways were rather similar for the two receptors, this means that the ligand-mediated signaling was responsible for the major part of the signaling capacity for ECRF3 (ligand-regulated/constitutive signaling ratio (L/C) > 1) and only a minor part for ORF74-HHV8 (L/C < 1), as most clearly seen in Fig. 10 (gray versus black bars) and in Table III. The lowest ligand contribution compared with constitutive activity was found for the G_i coupling of ORF74-HHV8 (L/C = 0.2). The G_i activity of ECRF3 (assessed by the cAMP inhibition experiments in HEK293 cells) (Fig. 8) resulted in a surprisingly low ligand regulation compared with the constitutive activity (L/C = 0.8). Thus, for this measurement (but not for the G_i component of SRE), the constitutive signaling slightly exceeded the ligand-regulated signaling (assuming that CXCL6/GCP-2 and vCCL2/vMIP-II are full agonist and full inverse agonist, respectively). This discrepancy in the ligand-regulated versus constitutive component of G_i signaling could be a reflection of the very different steps at which we measured in the G_i signaling cascade. Adenylate cyclase inhibition is close to the G-protein, whereas SRE is far away and influenced by several regulators from the G-protein to the nucleus. In addition, adenylate cyclase inhibition is dependent on pre-activation with forskolin. Since forskolin activates adenylate cyclase in all cells, the actual window of G_i activity is therefore dependent on transfection efficiency. In summary, the ligand-regulated signaling compared with the constitutive signaling was greater for ECRF3 than for ORF74-HHV8 in all cases. The ligand-regulated activities were also greater for ECRF3, whereas the constitutive activities were greater for ORF74-HHV8 (Fig. 10 and Table III).

What Are the Roles of Virus-encoded 7TM Chemokine Receptors?—Experimental evidence from receptor knockout studies indicates a certain importance of the chemokine receptors in the virus-host interplay. For instance, it has been shown that knockout of the murine cytomegalovirus-encoded receptor M33 results in a virus strain with decreased virulence and with less severe host infection (65). Similarly, deletion mutants of ORF74 from MHV68 point to a receptor function in the early reprogramming of the virus-infected cells and upon virus reactivation from latency (66, 67). Transgenic expression of ORF74-HHV8 in mice has shown that this receptor is important for Kaposi's sarcoma development (19, 20) as a side effect of high constitutive activity since abrogation of the constitutive activity decreases the appearance of Kaposi's sarcoma-like lesions (68).

In conclusion, virus-encoded chemokine receptors serve important functions, although they are not mandatory for virus survival. From a molecular pharmacology point of view, these receptors constitute a unique opportunity to study basic principles of ligand recognition, the activation mechanism of 7TM receptors, internalization, and recycling pathways as examples of targeted evolution, i.e. the receptors obtained from the host and through "combinatorial chemistry" structurally and functionally varied by random mutagenesis. The receptor phenotypes are highly interesting, with most of them being constitutively active. This study of ECRF3 addresses differences among virus-encoded chemokine receptors characterized so far with respect to ligand repertoire and signaling properties.

	FOOTNOTES

 
^* This work was supported by grants from the Danish Medical Council, the Danish Cancer Foundation, and the NovoNordisk Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This 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: Lab. for Molecular Pharmacology, Dept. of Pharmacology, Panum Inst., Bldg. 18.6, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Tel.: 45-3532-7608; Fax: 45-3532-7610; E-mail: rosenkilde{at}molpharm.dk.

¹ The abbreviations used are: 7TM, seven-transmembrane; PTx, pertussis toxin; ORF74, open reading frame 74; MHV68, murid -herpesvirus 68; HHV8, human herpesvirus 8; HVS, herpesvirus saimiri; CXCR, CXC chemokine receptor; IL-8, interleukin-8; NAP-2, neutrophil-activating peptide 2; IP10, interferon-inducible protein 10; GCP-2, granulocyte chemotactic protein 2; vCCL2, viral CCL2; vMIP-II, viral macrophage inflammatory protein II; SRE, serum response element; PI, phosphatidylinositol; NFAT, nuclear fact